Hybrid DIVEMA/PLGA nanoparticles as the potential drug delivery system

Hybrid DIVEMA/PLGA nanoparticles as the potential drug delivery system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hybrid DIVEMA/PLGA nanoparticles as the potential drug delivery system Marina Gorshkova, Lyudmila Vanchugova, Nadezhda Osipova, Alexey Nikitin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4594368/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The hybrid nanoparticles (NP) consisting of poly(lactic-co-glycolic acid) (PLGA) and polyanionic copolymer of divinyl ether with maleic anhydride (DIVEMA) were prepared by the high pressure homogenization – solvent evaporation technique or by nanoprecipitation and evaluated by physicochemical and spectroscopic methods. The nanoparticles formed by PLGA (MM 7–17 kDa) and DIVEMA (MM 20 kDa or 80 kDa) at mass ratios from 1.2:1 to 8:1 had the hydrodynamic diameter of ~ 200 nm, negative zeta potentials of -33 to -40 mV, and were stable upon freeze-drying. The presence of DIVEMA in the PLGA nanoparticles improved their properties as the drug carrier. Thus, loading of the model drug doxorubicin was increased 2-fold and its release time was considerably extended. The enhanced surface functionality of the hybrid nanoparticles was demonstrated by a ~ 5-fold higher content of the surface-conjugated PEGylated bovine serum albumin as compared with the plain PLGA nanoparticles. The DIVEMA/PLGA NP exhibited low cytotoxicity and good hemocompatibility. This is the first study that describes the DIVEMA/PLGA NP and demonstrates their potential as the drug delivery system. Hybrid nanoparticles Divinyl ether-co-maleic anhydride copolymer (DIVEMA) Doxorubicin Drug delivery systems PLGA Polyanion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The nanoparticles made of biodegradable and biocompatible copolymers of lactic and glycolic acids (PLGA) are widely used as nanocarriers for drug delivery [ 1 ]. However, similarly to other colloidal nanocarriers, the intravenously injected PLGA nanoparticles (PLGA NP) tend to accumulate in the organs of the mononuclear phagocyte system (MPS) that is specialized in the clearance of both endogenous and exogenous particulates from the blood. This biodistribution pattern is beneficial for drug delivery to macrophage-rich organs (such as liver or spleen) but interferes with delivery to other targets [ 2 ]. Moreover, the scarcity of the reactive groups on the surface of the PLGA NP, limited to the end carboxylic groups of PLGA, hampers attachment of bioligands that could enable receptor-mediated transport of such delivery systems to the targets outside the MPS. The commonly used solution of this problem is the employment of the nanoparticles composed of PEG-PLGA copolymers that have demonstrated effective avoidance of macrophages whereas the terminal groups of PEG moieties could be used for conjugation with the targeting bioligand. However, certain disadvantages of this technology, such as, for example, PEG immunogenicity inspired the search for alternative approaches [ 3 ]. Thus, one of the approaches involves modification of PLGA nanoparticles with polyelectrolytes, which, on one hand, increases hydrophilicity of their surface and on the other hand provides numerous functional groups for vectorization. Indeed, a number of studies are focused on fabrication of the PLGA nanoparticles modified with cationic polymers, which helps to improve their loading with nucleic acids (i.e. plasmid DNA) and proteins (i.e. antigens) by electrostatic interactions [ 4 – 6 ]. The drawback of the positively charged nanoparticles is that they are potentially more cytotoxic than negative or neutral ones, which is attributed to their stronger interaction with negatively charged cellular membranes that may cause the membrane damage [ 7 – 9 ]. Another option is modification of the nanoparticles with biocompatible polyanions, such as for example copolymers of maleic anhydride that proved to be suitable for many biomedical applications [ 10 ]. An attractive candidate for this approach is the copolymer of divinyl ether with maleic anhydride widely known as DIVEMA. This remarkable polymer extensively studied in the 70s and 80s exhibits the broad spectrum of biological activities such as induction of biological response to tumors, antiviral and antibacterial activity explained most probably by induction of interferon production and/or macrophage activation [ 11 , 12 ]. Due to this per se activity, DIVEMA became one of the first synthetic polymers clinically tested as the anticancer agent; however, despite the antitumor activity evidenced in numerous preclinical studies, no significant effect was observed in humans [ 13 , 14 ]. DIVEMA was synthesized in 1951 by G.B. Butler by radical copolymerization of maleic anhydride with divinyl ether yielding the copolymer with alternating structure (mole ratio of divinyl ether: maleic anhydride is 1:2) that contains the cycle developed from both monomers. This reaction was recognized as a new type of polymerization designated as “cyclocopolymerization” [ 15 ]. Initially, Butler assumed that the cycle had the tetrahydropyran structure, four atoms of which were contributed by the diene and two atoms by the maleic anhydride (that is why the copolymer became known also as Pyran copolymer) (Fig. 1). Further studies using 13 C-NMR suggested that the polymer chain could contain both tetrahydropyran and tetrahydrofuran cycles (Fig. 1) [12]. The results of Kunitake et al. indicated that the cycle structure could depend on the solvent polarity where the non-polar solvent favored the formation of six-membered cycles [16]. However later, Gorshkova et al. demonstrated that synthesis of DIVEMA in both chloroform (non-polar) and acetone (polar) led to formation of the polymer containing predominantly tetrahydrofuran cycles [17]. While the role of the cycle structure in the biological properties of DIVEMA has not been elucidated, both its activity and toxicity were shown to be dependent on its molecular mass (MM) and molecular mass distribution (MMD). Thus, the results of the preclinical and clinical studies exhibited the acceptable toxicity of the polymer with the MM of 15–20 kDa and narrow MMD (tolerable single intravenous dose in humans 2500 mg), whereas higher MM and wide MMD were associated with the increase of toxicity [ 12 , 13 ]. Although DIVEMA failed as the antitumor agent or immunomodulator, it may still be valuable for the purpose of drug delivery. Indeed, apart from its versatile biological activity, the advantages of DIVEMA, as the potential constituent of the nanoparticle-based delivery system, are its relatively low toxicity, facile synthesis, and abundant carboxylic groups (four carboxylic groups per each monomeric unit, Fig. 1) suitable for the surface modification. Therefore, the objective of the present study was to investigate the possibility to prepare the hybrid DIVEMA/PLGA nanoparticles and evaluate its potential as the drug delivery system. Considering the aforementioned toxicological data, the experiments were performed using the DIVEMA sample with MM of 20 kDa synthesized for this study. The sample with MM of 80 kDa was used in some experiments for comparison. 2. Materials and methods 2.1 Materials PLGA (RESOMER® RG 502H, lactide/glycolide = 50:50 mol/mol; acid terminated; MM 7,000–17,000 Da; η = 0.16–0.24 dL/g) was purchased from Evonik Röhm GmbH (Germany). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), N–hydroxysuccinimide (NHS), diisopropylethylamine (DIPEA), bovine serum albumin (BSA), polyvinyl alcohol (PVA, 9–10 kDa, 80% hydrolyzed), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (USA). 2-Iminothiolane hydrochloride (Traut's reagent) was purchased from ThermoFisher Scientific (USA). Maleimide PEG3500 Amine (MAL-PEG3500-NH 2 , TFA salt, MM 3500 Da) was from JenKem Technology (USA). The reactive derivatives of the fluorescent dyes cyanine5 (Cy5) amine and cyanine3 (Cy3) amine were purchased from Lumiprobe (Russia). All other chemicals were of analytical grade. 2.2 Synthesis and analysis of divinyl ether and maleic anhydride copolymer (DIVEMA) The DIVEMA copolymer with an alternating (1:2) structure was synthesized by radical copolymerization of divinyl ether and maleic anhydride as described previously in [ 17 ]. All reagents were thoroughly purified before the reaction: maleic anhydride was sublimated twice; divinyl ether was distilled over metallic Na; 2,2’-azobisisobutyronitrile (AIBN) used as an initiator was recrystallized twice from methanol and then dried in vacuum. The reaction was performed in the sealed glass ampoules in dry acetone that was purified and distilled immediately before use. Maleic anhydride and divinyl ether were added at the molar ratio of 2:1; the total monomer concentration was 1.28 mol/L; the AIBN concentration was 1.7 x 10 − 2 mol/L. The polymerization was performed at 60 °C. Tetrahydrofuran (THF, 22% v/v or 7% v/v) was added in the reaction mixture as the chain transfer agent. The resulting polymer was purified from low-molecular impurities by extraction in ether and then dried to a constant mass in vacuum. The molecular mass (MM) and mass distribution (MMD) of DIVEMA and DIVEMA-Cy3 conjugates were analyzed by gel permeation chromatography as described in [ 18 ] using the Gilson liquid chromatography system (Gilson Inc., USA) equipped with the refractive index and UV detectors and TSK-gel GMPW column (7.5 mm i.d. x 600 mm) with TSK guard PWH column (Tosoh Corp., Japan). Borate buffer (pH 10.2) with addition of 0.2M NaCl was used as the mobile phase; the elution rate was 1 mL/min. A series of poly(ethylene glycol)-poly(ethylene oxide) (PEG-PEO) standards with narrow MMD (Polymer Standards Service GmbH, Germany) was used to obtain a calibration curve that was a straight line for molecular masses in the range of 1.0×10 3 –1.2×10 6 Da. The intrinsic viscosity was measured at 25 °C in the same borate buffer using the Ubbelohde suspended-level type capillary viscometer (Canon, England). 2.3. Preparation of DIVEMA/PLGA nanoparticles The nanoparticles were prepared by the homogenization – solvent evaporation technique or by nanoprecipitation. In the homogenization – solvent evaporation technique, DIVEMA and PLGA were used in the mass ratio of ~ 1:8. The solutions of PLGA (600 mg, in 7.2 mL of dichloromethane [DCM]) and DIVEMA (72 mg in 4.8 mL of acetone) were mixed and this organic phase was added to 60 mL of a 0.5% aqueous solution of polyvinyl alcohol (PVA, 9–10 kDa) used as surfactant. The mixture was first emulsified using the high-shear homogenizer (Ultra-Turrax T18, IKA, Germany; 23600 rpm), and then this coarse emulsion was further processed using the high-pressure homogenizer (Microfluidics M-110P, Microfluidics, USA; 15000 psi). Then the organic solvent was removed under vacuum; the resulting suspension was separated from residual DIVEMA by centrifugation and thorough washing with water and then freeze-dried with 2.5% (w/v) of D-mannitol as cryoprotectant. The plain PLGA nanoparticles were obtained in a similar way; in this case, 4.8 mL of acetone without DIVEMA was added to the polymer solution in DCM. In the case of nanoprecipitation, the solution of PLGA (60 mg) and DIVEMA (50 mg, 23 mg, or 7.5 mg for DIVEMA/PLGA mass ratios of 1:1.2, 1:2.6, 1:8, respectively) in 6 mL of the ACN - acetone mixture (1:2 v/v) was added dropwise to 60 mL of a 0.5% aqueous PVA solution under stirring and incubated for 3 h. The resulting nanosuspension was processed and freeze-dried as described above. The plain PLGA nanoparticles were obtained similarly without addition of DIVEMA. 2.4. Preparation of fluorescently labeled nanoparticles for fluorescence spectroscopy study DIVEMA (MM 20 kDa) was labeled with the Cyanine3 dye (Cy3, λ ex 555 nm, λ em 570 nm) by covalent binding of its water-soluble amine derivative (Cy3 amine) to carboxylic groups via the NHS/DCC coupling reaction. The solution of N-hydroxysuccinimide (NHS, 23 mg in 1 mL of acetone) was added to the DIVEMA solution (600 mg in 30 mL of acetone), and the mixture was stirred for 30 min in the dark. Then N,N'-dicyclohexylcarbodiimide (DCC, 14 mg in 0.5 mL of acetone) and Cy3 amine (18 mg in 1.5 mL of acetone-EtOH mixture) were added, and the mixture was stirred for another 3 h. Then the polymer was precipitated into the 10-fold volume of ether; the precipitate was separated by filtration, washed with chloroform and dried in vacuum. The polymer precipitation procedure was repeated three times. The absence of the unbound dye was monitored by the absence of absorption at 543 nm in the UV spectra of the washing solvent. The dye content in the DIVEMA-Cy3 conjugate was determined by UV spectroscopy (λ max 552 nm). The conjugate containing 2.7% w/w of the dye (dye-to-polymer ratio of 1:40, w/w) was used in the experiments. The DIVEMA-Cy3 conjugate was also analyzed by gel permeation chromatography as described above using the refractive index detector and the UV detector. PLGA was labeled with the Cyanine5 dye (Cy5, λ ex 651 nm, λ em 670 nm) by covalent binding of its water-soluble derivative Cy5 amine to the terminal carboxylic groups of PLGA via the NHS/EDC coupling reaction as described earlier [ 19 , 20 ]. Briefly, solutions of Cy5 amine, DIPEA, EDC, and NHS in DCM were added to the PLGA solution in DCM. The reaction mixture was incubated for 48 h under continuous stirring at room temperature in the dark. The obtained solution was washed 3 times with water and water/methanol (1:1) mixture. The organic phase was separated and dried over anhydrous sodium sulfate with subsequent evaporation. The obtained sediment was dissolved in ethyl acetate, and then added to the tenfold volume of hexane to precipitate the polymer. The precipitate was dried in vacuum. The content of Cy5 in the conjugate was measured spectrophotometrically. The PLGA-Cy5 conjugate with a dye-to-polymer ratio of 1: 600 (w/w) was used in the experiments. The PLGA-Cy5 conjugate was analysed by gel permeation chromatography using the Waters HPLC system equipped with a set of Styrogel HR5E and HR4E columns (300 mm × 7.8 mm). Tetrahydrofuran was used as a solvent and eluent at the flow rate of 1.00 mL/min. Polymer solutions were prepared at the known concentration (ca. 1 mg/mL), injection volume was 50 µl. Data were collected using the refractive index detector (Waters 2414 RI Detector) and the UV detector (Milton Roy UV-detector 3100 model, λ 264 nm). Data analysis was performed with the Z-lab software. The system was calibrated using the polystyrene standards set. Formation of the PLGA-Cy5 conjugate was also confirmed by the TLC method on silica gel-coated plates using the DCM/methanol/water (6.5:2.5:0.4 v/v) mixture as eluent. The dual-labeled DIVEMA-Cy3/PLGA-Cy5 nanoparticles were prepared by nanoprecipitation as described above using the DIVEMA/PLGA mass ratio of 1:2.6. The 1:1 mixture of the PLGA-Cy5 conjugate and non-modified PLGA was used as the PLGA constituent. The fluorescence spectra of the labeled nanoparticles were registered using the RF-6000 spectrofluorometer (Shimadzu, Japan). 2.4. Physicochemical characterization of nanoparticles The average hydrodynamic diameter (ZaveD) and polydispersity index (PDI) of the nanoparticles were determined by the dynamic light scattering method (DLS) using the Zetasizer Nano ZS instrument (helium-neon laser source with a wavelength of 632.8 nm, light scattering angle 173°, temperature 25°C; Malvern Instruments, UK, Great Britain). For measurements, the freeze-dried samples were resuspended in water and then the suspensions were diluted 50-fold with water. The zeta potential was measured by electrophoretic light scattering using the same instrument. Each measurement was repeated 4 times. The content of carboxylic groups on the nanoparticles or in the supernatants after nanoparticle separation by centrifugation was determined by potentiometric titration using the pH-meter with combined microelectrode. The Fourier transform infrared spectroscopy (FTIR) study was performed using the PerkinElmer Spectrum One FTIR spectrometer equipped with the universal ATR Sampling Accessories with Diamond/ZnSe crystal (PerkinElmer Inc., USA). 2.5. Analysis of nanoparticle morphology by microscopy The scanning electron microscopy (SEM) study of the nanoparticles was carried out using the JSM 6510 LV scanning electron microscope (Jeol Ltd., Japan) in the secondary electron mode. To improve the image quality the nanoparticles were washed from mannitol by repeated centrifugation. For this, the freeze-dried samples were reconstituted to the initial volume with distilled water, and then the suspension was subjected to ultrasonication and centrifuged at 20000 rpm for 30 min. This procedure was repeated 3 times using the same volume of water. Then the drop of the suspension was placed on the substrate, dried in air, sputtered with platinum for 30 s, and placed in the microscope. The DIVEMA/PLGA nanoparticles used for the SEM study were prepared by the homogenization – solvent evaporation technique using DIVEMA with MM of 20 kDa, the PLGA : DIVEMA mass ratio was 8:1. The similarly prepared plain PLGA NP were used for comparison. 2.6. Synthesis of FITC-labeled bovine serum albumin (FITC-BSA) The fluorescein isothiocyanate (FITC) solution in DMSO (0.0026 M, 1 mL) was added dropwise to the bovine serum albumin (BSA) solution in freshly prepared 0.1 M NaHCO 3 /NaOH buffer (100 mg, 10 mL, pH 9). The BSA-FITC molar ratio was 1:2. The mixture was incubated under stirring at room temperature for 1 h and then for 15 h at 5–6°C in the dark. Then the ammonium chloride was added into this solution to the concentration of 0.05 M, and stirring was continued for 1 h. The labeled protein was separated from free dye (FITC) by gel permeation chromatography using a Sephadex G-25 column (eluent – 0.1 M phosphate buffer, pH 7.4). The purification procedure was repeated twice. The resulting FITC-BSA solution was freeze-dried without addition of a cryoprotectant. The FITC-BSA content and the FITC/protein (F/P) ratio were determined by spectrophotometry. To determine the content of FITC-BSA and the F/P ratio the FITC-BSA sample was dissolved in water and diluted 5-fold. The absorbance was measured at 494 nm and 280 nm. The protein concentration was calculated as: $$\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n} \text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} \left(\text{M}\right)= \frac{{(\text{A}}_{280}-{\text{A}}_{494})\times \text{C}\text{F}(\text{F}\text{I}\text{T}\text{C})}{{\epsilon }\left(\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\right)} \times \text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n}$$ $$\text{c}\left(\text{B}\text{S}\text{A}\right) \left(\text{M}\right)= \frac{{(\text{A}}_{280}-{\text{A}}_{494})\times 0.3}{43824} \times \text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n}$$ $$\text{F}/\text{P}= \frac{{\text{A}}_{494}}{68000 \times \text{C}\left(\text{B}\text{S}\text{A}\right)}$$ The FITC-BSA concentration in this sample was 0.83 mg/mg lyophilisate, F/P = 1.08. 2.7. Conjugation of DIVEMA/PLGA NP with FITC-BSA via PEG linker Thiolation of FITC-BSA using Traut’s reagent . The FITC-BSA solution (30 mg in 1.0 mL of PBS buffer, pH 8.0) was mixed with the Traut’s reagent solution (200 µg in 0.1 mL MilliQ water). The mixture was incubated for 1 h at 20°С under stirring (500 rpm). Then the unbound reagent was removed by filtration through the Microcon 30 kDa filter. The thiolated protein (FITC-BSA-SH) was separated from the unbound dye and other low molecular impurities by repeated ultrafiltration using the Microcon 30 kDa membrane filter (centrifugation at 20 000 rpm, 5°C, 20–30 min), and then the protein solution was diluted to 2.0 mL with MQ water and freeze-dried. PEGylation of FITC-BSA-SH using maleimide-PEG3500-amine . The FITC-BSA-SH solution (10 mg in 0.5 mL PBS, pH 7.4) was mixed with the MAL-PEG3500-NH 2 solution (3.0 mg of in 0.5 mL PBS, pH 7.4). The mixture was incubated for 48 h at 4°С, 500 rpm. The FITC-BSA-PEG-NH 2 fraction was purified by repeated ultrafiltration using the Microcon 30 kDa membrane filter, then diluted with water to 2.0 mL and freeze-dried. Activation of DIVEMA/PLGA nanoparticles carboxylic groups using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). 14.0 mg of the freeze-dried nanoparticles (7.5 µM of –COOH groups) was resuspended in 0.3 mL of MQ water, then 0.72 mg (3.9 µM) of EDC in 0.1 mL of MQ water and 1.75 mg (15.2 µM) of NHS in 0,1 mL of MQ were added. The mixture was incubated for 3 h at 20°С under stirring. Conjugation of DIVEMA/PLGA NP with FITC-BSA-PEG-NH 2 . The FITC-BSA-PEG-NH 2 solution (5.0 mg in 0.5 mL of MQ water) was added to the activated NP and incubated for 12 h at 20°С under stirring. The unbound FITC-BSA-PEG-NH 2 was removed by centrifugation (20 000 rpm, 5°C, 20–30 min) and purified by repeated ultrafiltration using the Microcon 30 kDa filter as described above. The nanoparticles were resuspended in 2.0 mL of MQ water using the US-bath and Vortex and then freeze-dried with 1% (w/v) of D-mannitol as a cryoprotectant. Alternatively, the surface of the DIVEMA/PLGA NP was modified with FITC-BSA-PEG by adsorption. Conjugation of FITC-BSA-PEG-NH 2 with the similarly activated plain PLGA NP was performed as described above. 2.8. Preparation of doxorubicin-loaded DIVEMA/PLGA nanoparticles and evaluation of doxorubicin release rate The DIVEMA/PLGA nanoparticles loaded with doxorubicin (DIVEMA/PLGA-Dox NP) were obtained by the high pressure homogenization - solvent evaporation technique (w/o/w double emulsion method). PLGA (600 mg in 3.6 mL of DCM) and DIVEMA (60 mg in 2.4 mL of acetone) were mixed, and then this organic phase was added to the solution of doxorubicin in 0.001 N HCl (30 mg in 4.7 mL). The mixture was emulsified using the disperser (Ultra-Turrax T18, 23600 rpm). This primary emulsion was added to the 1% PVA solution in phosphate-buffered saline (30 mL). The mixture was first emulsified using the Ultra-Turrax and then using the high-pressure homogenizer as described above. Thereafter, the organic solvent was removed under vacuum and then the suspension was lyophilized with the addition of 5% (w/v) D-mannitol as a cryoprotectant. When preparing the plain PLGA NP loaded with doxorubicin (PLGA-Dox NP), the polymer was dissolved in 6 mL of DCM. The total content of doxorubicin was determined spectrophotometrically (λ max 480 nm) after dissolution of the freeze-dried nanoparticles in DMSO. The encapsulation efficiency of doxorubicin was determined after separation of the nanoparticles by ultrafiltration using Amicon® Ultra-0.5 filters (Millipore, MWCO 100 kDa) and subsequent spectrophotometric measurement of the free drug concentration in the filtrate. The doxorubicin release rate from the DIVEMA/PLGA-Dox NP and PLGA-Dox NP was evaluated using the 0.9% sodium chloride solution as a physiologically relevant medium. The freeze-dried nanoparticles were resuspended in 25 mL of 0.9% sodium chloride (total doxorubicin concentration 65–68 µg/mL) and placed in the shaker-incubator (200 rpm, 37°C). The samples were taken after 0, 1, 2, 3, 4, 6, and 24 h of incubation, and then the nanoparticles were separated by centrifugation (48300 g, 18°C, 30 min, Avanti JXN-30, Beckman Coulter, USA). The doxorubicin concentration in the supernatants was determined spectrophotometrically (λ max 480 nm). For each sample, 3 parallel measurements were carried out. 2.9. Evaluation of DIVEMA/PLGA NP cytotoxicity The porcine kidney cells (LLC-PK1) and human hepatocellular carcinoma cells (Hep G2) were used for testing of the nanoparticle cytotoxicity in vitro . The cell lines were obtained from the American Type Culture Collection (ATCC, USA). The cell viability was assessed using the MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma, USA) reduction assay. The test was conducted according to the manufacturer’s protocol. Briefly, MTT was dissolved in PBS at 5 mg/mL (stock solution). The cells were cultured in a humid atmosphere (37°C, 5% CO 2 , 95% air) using DMEM (Gibko) media containing 10% FBS (Biowest, USA) with the addition of a mixture of antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, Gibco, USA), GlutaMAX (2 mM, Gibco, USA). The cells were seeded in a 96-well plate (1×10 4 cells/well), cultured for 24 h, and then incubated with different amounts of the nanoparticles in the range of 0-100 µg/mL. After 24-h incubation with the samples, the MTT stock solution (10 µl per 100 µl medium) was added to all wells, and the cells were incubated for another 4 hours at 37° C. Dimethylsufloxide (DMSO; 200 µl) was added to each well after removal of the supernatant to dissolve the dark blue crystals. After shaking the plate for 10 min, the cell viability was assessed by measuring the absorbance at 570 nm using the microplate scanning spectrophotometer; all measurements were performed three times. The results are represented as the average of three independent experiments. 2.10. Evaluation of DIVEMA/PLGA NP hemocompatibility The experiments involving manipulations with human whole blood or blood plasma were approved by the local ethics committee of the N.N. Burdenko National Medical Research Center of Neurosurgery (Moscow, Russia; approval No. 11/2020). The hemolytic effects of the nanoparticles and their influence on coagulation of human blood plasma were assessed as described previously [ 21 ]. The hemolytic effect of the nanoparticles was analyzed by the in vitro colorimetric assay. Briefly, the DIVEMA/PLGA NP were added to the blood samples (human blood obtained from healthy volunteers) in the concentration of 100 µg/mL and incubated for 3 hours at 37°C. To determine total hemoglobin (tHb), 1% Triton X-100 was added to a separate blood sample to induce hemolysis (positive control). After incubation, the undamaged human red blood cells (RBC) were separated by centrifugation. The supernatants were transferred into 96-well plates. Ferricyanide oxidizes oxyhemoglobin to methemoglobin, and cyanide converts methemoglobin to cyanmethemoglobin, a red-colored derivative of hemoglobin, that is detected by spectrophotometry (λ max 540 nm). The acute in vitro hemolytic properties of the nanoparticles were evaluated as the percent of hemolysis calculated as the ratio of the optical density of the sample to the optical density of the control. The influence of the DIVEMA/PLGA NP and PLGA NP on coagulation of human blood plasma (i.e. blood clotting) was evaluated by measurement of the prothrombin time (PT) after plasma incubation with the nanoparticles. The nanoparticles were added to the blood samples in the concentrations of 1, 10, and 100 µg/mL. The platelet-poor plasma was isolated by centrifugation (1500 g, 10 min, 25°C). The blood samples were incubated with the nanoparticles for 30 min at 37°C under continuous stirring and placed into the coagulometer. Then the calcium thromboplastin was added to activate the clot formation followed by the measurement of coagulation time. Measurements were performed in triplicates. The nanoparticle effect on human peripheral blood platelet aggregation was evaluated by flow cytometry as described in [ 21 ]. This approach is based on the measurement of the platelet activation level as a percentage of P-selectin expression on platelet surface. In brief, the platelet-rich plasma (PRP) was obtained by centrifugation (1000 rpm, 10 min) and incubated with nanoparticles in the concentrations of 1-100 µg/mL. The aggregation inductor ADP (adenosine diphosphate) was added to the positive control samples. Platelets were then stained by fluorescently-labeled anti-CD36 antibodies (FITC-conjugated); the subpopulation of activated platelets was determined using anti-CD36 and PE-conjugated anti-CD62 antibodies (indicate the level of P-selectin, Biolegend, USA). The samples were analyzed by flow cytometry. 3. Results and discussion DIVEMA was synthesized by radical cyclopolymerization of divinyl ether and maleic anhydride in dry acetone with AIBN used as the initiator. As shown previously by Gorshkova et al., this process led to formation of the copolymer with the tetrahydrofuran cycles which was confirmed by 13 C-NMR (data not shown) [ 17 ]. As mentioned above, the acceptable toxicity of DIVEMA was observed at the relatively low molecular mass of 18–20 kDa and narrow MMD. Therefore, to enable the MM control THF was added in the reaction mixture as the chain transfer agent. The molecular mass parameters of the polymers obtained at different THF concentrations were analyzed by GPC as described in [ 18 ]. The copolymers obtained in the presence of 22% v/v or 7% v/v of THF had the molecular masses of 20 kDa and 80 kDa, respectively. For comparison, in the absence of THF the copolymer MM reached 220 kDa (Table 1 ). Table 1 Molecular mass characteristics of DIVEMA samples synthesized in the presence of THF THF content, % v/v DIVEMA molecular mass characteristics, Da [η], g/dl MM Mn Mw/Mn 0 220000 56000 3.9 0.81 7 80000 29600 2.7 0.41 22 20000 12100 1.66 0.16 The PLGA NP are usually prepared by the high-pressure homogenization – solvent evaporation technique or by nanoprecipitation [ 22 ]. In the present study, the DIVEMA/PLGA NP were prepared by both techniques using the mixture of DIVEMA (MM 20 kDa or 80 kDa) and PLGA; polyvinyl alcohol (0.5% aqueous solution, PVA MM 9–10 kDa) was used as surfactant in both cases. The physicochemical parameters of the DIVEMA/PLGA NP and plain PLGA NP obtained by both methods at different DIVEMA/PLGA ratios are shown in Table 2 . Both methods yielded the hybrid nanoparticles with similar hydrodynamic diameters of ~ 200 nm; the results were fairly reproducible (RSD % <15%). The nanoparticles obtained by high pressure homogenization had higher polydispersity, which is probably due to a higher initial concentration of PLGA used in this method. The molecular mass of DIVEMA did not exert any influence on the size of nanoparticles produced by nanoprecipitation: the nanoparticles produced using DIVEMA with MM of 20 kDa and 80 kDa had similar sizes of ~ 200 nm. However, as compared with the plain PLGA NP, the DIVEMA/PLGA NP exhibited considerably bigger average hydrodynamic diameters, which suggests the presence of the more pronounced hydration shell. Accordingly, the content of the –COOH groups in the DIVEMA/PLGA NP was considerably higher as compared to the plain PLGA NP (0.74 ± 0.08 vs 0.09 ± 0.02 mmol/g PLGA; DIVEMA/PLGA = 1:8) indicating the presence of abundant polyanionic groups on the surface of the hybrid nanoparticles. Table 2 Physicochemical parameters of DIVEMA/PLGA NP (mean ± SD, n = 5) Method of preparation DIVEMA/ PLGA ratio, mg/mg Nanoparticle size and size distribution (DLS) Zeta potential, mV Average size, nm Polydispersity index (PDI) DIVEMA 20 kDa High pressure homogenization 1:8 243 ± 26 0.205 ± 0.028 -35.5 ± 3.0 0:1 180 ± 25 0.240 ± 0.090 -20.3 ± 1.7 Nanoprecipitation 1:8 204 ± 24 0.113 ± 0.028 -33.0 ± 2.0 1:2.6 194 ± 1 0.081 ± 0.014 -35.7 ± 0.4 1:1.2 221 ± 3 0.106 ± 0.009 -36.4 ± 1.6 0:1 126 ± 14 0.081 ± 0.012 -17.5 ± 3.9 DIVEMA 80 kDa Nanoprecipitation 1:8 176 ± 2 0.131 ± 0.015 -39.7 ± 1.6 1:2.6 222 ± 3 0.105 ± 0.026 -34.7 ± 0.7 The nanoparticle morphology was investigated by scanning electron microscopy (SEM). For better image quality, the nanoparticles were washed form mannitol and coated with platinum. According to the DLS measurements, the DIVEMA/PLGA NP used for the SEM study had the hydrodynamic diameter of 203 nm (PDI 0.20). The plain PLGA NP were slightly smaller (mean diameter 180 nm, PDI 0.16). As seen from Fig. 2, both hybrid and plain nanoparticles had the spherical shape. Precise comparison of the size measurements obtained by SEM and DLS is not possible due to the high polydispersity of the nanoparticles; however, it appears that in the SEM micrographs the majority of the hybrid nanoparticles had smaller diameters than the diameter measured by DLS (100–150 nm vs 203 nm). At the same time, in the case of the plain PLGA NP both methods yielded very similar results. This phenomenon may be attributed to the loss of the more considerable hydration shell of the DIVEMA/PLGA NP that was lost during drying. FTIR spectroscopy study. The presence of DIVEMA in the DIVEMA/PLGA NP was confirmed using the FTIR spectroscopy. The sample of the thoroughly washed and freeze-dried DIVEMA/PLGA NP with the high content of DIVEMA (DIVEMA/PLGA mass ratio = 1:1.2, DIVEMA MM 20 kDa) was used in the FTIR study to achieve better interpretation of the spectra. The shift of the PLGA carbonyl bond from 1746.77 cm -1 to a higher frequency and its broadening in the spectrum of the DIVEMA/PLGA NP suggests the contribution of the DIVEMA`s carbonyls into this bond (Fig. 3 ) and is also due to the formation of H-bonds [ 23 ]. This shift became more considerable with the increase of the DIVEMA content in the nanoparticles (data not shown). Moreover, the increase of the DIVEMA content led to the decrease of the PLGA carbonyl band intensity and the simultaneous increase in the anhydride band area and intensity (Table 3 ). Appearance of additional bonds corresponding to the anhydride cycle (1759.79 cm -1 ) and carbonyl groups of hydrolyzed DIVEMA (1713.08 cm -1 ) was also observed in the differential spectra of the DIVEMA/PLGA NP (Fig. 4 , Table 3 ). Table 3 Parameters of carbonyl and anhydride cycle bonds in the FTIR spectra of PLGA NP and DIVEMA/PLGA NP (DIVEMA/PLGA mass ratio = 1:1.2, DIVEMA MM 20 kDa) Nanoparticle type DIVEMA/ PLGA ratio, mg/mg Band parameters D1750/ D1380 carbonyl band, normalized spectra Anhydride cycle band at 1759 cm − 1 , differential spectrum Band intensity Band area, cm − 1 D1759 band intensity A1759 band area, cm − 1 PLGA 0:1 4.4 184.2 - - DIVEMA/ PLGA NP 1:25 4.1 182.7 0.081 2.74 DIVEMA/ PLGA NP 1:1.2 3.8 173.6 0.139 5.45 Furthermore, the presence of the DIVEMA carboxylic groups on the nanoparticle surface was also confirmed by the dependence of the nanoparticle size measured by DLS on the pH and ionic strengths of the media. Thus, in contrast to the plain PLGA NP that maintained their size in the pH range of 3 to 6, the average size (DLS) of the hybrid nanoparticles, obtained by both nanoprecipitation and homogenization significantly depended on pH decreasing from 221 nm and 256 nm at pH 6.0 to 125 nm and 178 nm, respectively, at pH 3.0 (Fig. 5). This phenomenon is most probably explained by the decreased dissociation of DIVEMA carboxylic groups at lower pH level. The pH-dependent size of the polyelectrolyte-coated nanoparticles was also observed by other authors [ 24 ]. Similar decrease of the DIVEMA/PLGA NP’s size observed in the 0.9% NaCl solution is due to the known coiling of polyelectrolytes in the presence of salts [ 25 ]. Fluorescence spectroscopy study. The hybrid structure of the DIVEMA/PLGA NP was confirmed explicitly by the fluorescence analysis of the hybrid nanoparticles composed from the polymers labeled with the fluorescent dyes: PLGA was labeled with the Cyanine5 dye (Cy5, λ ex 646 nm, λ em 662 nm) and DIVEMA was labeled with the Cyanine3 dye (Cy3, λ ex 555 nm, λ em 570 nm). The choice of these dyes is based on their known donor - acceptor properties. This dye pair exhibits the distance-dependent Förster resonance energy transfer (FRET) phenomenon that appears only when both dye molecules are localized in the close proximity [ 26 ]. The water-soluble amine derivatives of the fluorescent dyes were bound covalently to the carboxylic groups of the polymers using the NHS/carbodiimide reactions. The covalent attachment of the dyes to the polymers was confirmed by the GPC analysis as described above in [ 19 , 27 ]. The chromatograms of the labeled and non-labeled polymers obtained using the UV detector are shown in Fig. 6. The dual-labeled DIVEMA-Cy3/PLGA-Cy5 NP were prepared by nanoprecipitation using the PLGA-Cy5 conjugate (1:1 mixture of the conjugate and non-modified PLGA) and DIVEMA conjugate with the Cy3. The physicochemical parameters of the DIVEMA-Cy3/PLGA-Cy5 NP were similar to that of the non-labeled nanoparticles (Table 4 ). Table 4 Physicochemical parameters of fluorescently labeled nanoparticles obtained by nanoprecipitation (DIVEMA MM 20 kDa, DIVEMA/PLGA mass ratio = 1:2.6) Nanoparticle type Nanoparticle size and size distribution (DLS) Zeta potential, mV Average size, nm Polydispersity index (PDI) DIVEMA-Cy3/PLGA-Cy5 193 ± 2 0.040 ± 0.012 -25.1 ± 1.0 DIVEMA-Cy3/PLGA 194 ± 2 0.079 ± 0.008 -19.9 ± 0.3 DIVEMA/PLGA 194 ± 1 0.081 ± 0.023 − 35.7 ± 0.4 Comparison of the DIVEMA-Cy3/PLGA-Cy5 NP fluorescence spectra registered at λ ex 530 nm and 630 nm and the DIVEMA-Cy3/PLGA NP spectrum registered at λ ex 530 nm revealed the decrease of the fluorescence intensity of Cy3 (donor) in parallel with the significant increase in the fluorescence intensity of Cy5 (acceptor) for the dual-labeled nanoparticles at λ ex 530 nm (Fig. 7 ). Together with the known stable retention of the fluorescence labels, the FRET phenomenon observed for the dual-labeled nanoparticles is the strong argument in favor of the close proximity of two polymers and stability of the hybrid particles in aqueous media. Altogether this system stability suggests the interaction between DIVEMA and PLGA that is most probably explained by the formation of H-bonds between DIVEMA hydroxyl and carbonyl groups and PLGA ester groups. Enhanced surface functionality . The enhanced functionality of the DIVEMA/PLGA NP was demonstrated by the effective conjugation of the FITC-labelled PEGylated bovine serum albumin (FITC-PEG-BSA) chosen as the model biovector. The COOH-enriched nanoparticle surface enabled conjugation of 550 µg of protein per 1 mg of the nanoparticles, which is > 100-fold higher than the content of 4 µg/mg NP achieved by the protein conjugation with the end carboxylic groups of PLGA in the plain PLGA NP (Table 5 ). The latter result correlates with the data of other authors. The adsorption of FITC-BSA on the DIVEMA/PLGA NP was considerably less effective (only 118 µg/mg NP). Table 5 Physicochemical parameters of DIVEMA/PLGA NP modified with FITC-BSA-PEG (representative data) Method of FITC-BSA-PEG attachment Nanoparticle type FITC-BSA-PEG content, µg/mg NP Average NP size (DLS), nm Polydispersity index (PDI) Zeta-potential, mV Conjugation DIVEMA/PLGA 550 278 ± 6 0.189 ± 0.033 -30.0 ± 1.5 Adsorption DIVEMA/PLGA 118 267 ± 4 0.175 ± 0.028 -31.0 ± 1.2 Conjugation PLGA ~ 4 180 ± 2 0.150 ± 0.017 -25.0 ± 1.6 Prolongation of the drug release rate . Another potentially useful feature of the DIVEMA/PLGA NP such as the possibility to optimize the drug encapsulation and release rate was demonstrated using the antitumor antibiotic doxorubicin as the model drug. Indeed, the presence of DIVEMA led to a very considerable increase of the doxorubicin encapsulation efficiency in the hybrid nanoparticles as compared to the plain PLGA NP (97.5% and 50.7% for the DIVEMA/PLGA NP and PLGA NP, respectively, Table 6 ). Furthermore, the presence of DIVEMA also prolonged the drug release rate from the nanoparticles: approximately 20% and 50% of the drug was released from the DIVEMA/PLGA NP during the first 4 h and 24 h of the experiment, while the PLGA NP released ∼60% and ∼90% of the drug during the same time periods (Fig. 9). The influence of DIVEMA on the doxorubicin loading and release rate is most probably enabled by the electrostatic interaction between its numerous carboxylic groups and the amine group of doxorubicin as described earlier [ 28 ]. Table 6 Physicochemical parameters of doxorubicin-loaded DIVEMA/PLGA-Dox and PLGA-Dox nanoparticles (representative data) DIVEMA/PLGA/ Dox, mg/mg Total drug content, mg/mL Encapsulation efficiency of doxorubicin, % Average NP size, nm Polydispersity index (PDI) Zeta-potential, mV 0: 20:1 0.73 56.2 133 ± 1 0.214 ± 0.007 -1.8 ± 0.5 2: 20:1 0.69 95.5 203 ± 4 0.225 ± 0.019 -6.5 ± 0.8 Evaluation of cytotoxicity and hemocompatibility . The cytotoxicity and hemocompatibility of the DIVEMA/PLGA NP were assessed using the samples containing DIVEMA with MM 20 кDa or 80 kDa obtained by the nanoprecipitation method (DIVEMA/PLGA ratio = 1:8, Table 1 ). The plain PLGA NP were used as control. The cytotoxicity was assessed in vitro in the porcine kidney epithelial cells LLC-PK1 and Hep G2 human hepatocellular carcinoma cells by the МТТ-test. The reduction of viability of both cell types after incubation with the DIVEMA/PLGA NP or plain PLGA NP in the concentration of up to 100 µg/mL did not exceed 20%, which is generally accepted as the non-toxic effect [ 29 ]. The hematotoxicity was evaluated by the generally accepted tests, such as the level of hemolysis, blood coagulation test, and platelet activation test as described earlier [ 21 , 30 , 31 ]. Both types of nanoparticles did not produce any significant influence on blood components. Thus, the hemolysis index determined as the percentage of positive control (blood samples after incubation with Triton X100) was found to be less than 2% for all samples, which is classified as “non-hemolytic” according to the NCL nanoparticle characterization protocols established by the US National Cancer Institute [ 32 ]. The DIVEMA/PLGA NP as well as the plain PLGA NP used as control did not influence the blood coagulation time at concentrations of up to 100 µg/mL: the prothrombin time (PT) values remained within the normal physiological range (12–15 s) for all nanoparticle types; no statistically significant difference was observed between the nanoparticles. Incubation of platelet-rich plasma with both nanoparticle types did not induce platelet activation in all tested concentrations up to 100 µg/mL. The percent of the CD62P-positive cells used as a platelet activation marker did not significantly differ from control values. Low cytotoxicity and good hemocompatibility of the PLGA NP correlated with the previously reported results [ 21 ]. 4. Conclusion The hybrid DIVEMA/PLGA nanoparticles appear to be the stable nanosystem that exhibits important advantages as compared to the plain PLGA nanoparticles. Due to the enhanced surface functionality this nanosystem offers not only the improved binding of biovectors but also the optimized drug carrier properties which will be useful for many drugs capable of electrostatic interaction with the carboxylic groups of DIVEMA. Taken together with low cytotoxicity and good hemocompatibility of the hybrid nanoparticles, these properties suggest that they could be considered as a promising and versatile nanocarrier. This is the first study that describes the DIVEMA/PLGA NP and demonstrates their potential as the drug delivery system. Declarations Conflict of interest The authors declare no conflict of interest Funding The study was fulfilled with the financial support from the Ministry of Science and Higher Education of the Russian Federation (state assignment project FSSM-2022-0003). Author Contribution M.G. and S.G. conceptualized the study. L.V., N.O., A.N., J.K., E.K., Yu.E., J.M., T.K., conducted the experiments; J.K. prepared all figures; M.G., T.K. and S.G. performed the data analysis and wrote the manuscript text. All authors have read and agreed to the published version of the manuscript. Acknowledgement The study was carried out within the framework of the State Program of TIPS RAS. The authors are grateful to the D. I. Mendeleev Center for Collective Use of Scientific Equipment for performing the analytical tests. References Rezvantalab S, Drude NI, Moraveji MK et al (2018) PLGA-based nanoparticles in cancer treatment. Front Pharmacol 9:1260. https://doi.org/10.3389/fphar.2018.01260 Golombek SK, May JN, Theek B et al (2018) Tumor targeting via EPR: Strategies to enhance patient responses. Adv Drug Deliv Rev 130:17–38. https://doi.org/10.1016/j.addr.2018.07.007 Schubert J, Chanana M (2018) Coating Matters: Review on Colloidal Stability of Nanoparticles with Biocompatible Coatings in Biological Media, Living Cells and Organisms. Curr Med Chem 25:4553–4586. https://doi.org/10.2174/0929867325666180601101859 Soler Besumbes E, Fornaguera C, Monge M et al (2019) PLGA cationic nanoparticles, obtained from nano-emulsion templating, as potential DNA vaccines. Eur Polym J 120:109229. https://doi.org/10.1016/j.eurpolymj.2019.109229 Wusiman A, Gu P, Liu Z et al (2019) Cationic polymer modified PLGA nanoparticles encapsulating alhagi honey polysaccharides as a vaccine delivery system for ovalbumin to improve immune responses. Int J Nanomed 14:3221–3234. https://doi.org/10.2147/IJN.S203072 Tracey SR, Smyth P, Herron UM et al (2023) Development of a cationic polyethyleneimine-poly(lactic-co-glycolic acid) nanoparticle system for enhanced intracellular delivery of biologics. RSC Adv 13:33721–33735. https://doi.org/10.1039/d3ra06050k Fröhlich E (2012) The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomed 7:5577–5591. https://doi.org/10.2147/IJN.S36111 Lin HP, Akimoto J, Li YK, Ito Y (2020) Selective Control of Cell Activity with Hydrophilic Polymer-Covered Cationic Nanoparticles. Macromol Biosci 20:2000049. https://doi.org/10.1002/mabi.202000049 Li S, Malmstadt N (2013) Deformation and poration of lipid bilayer membranes by cationic nanoparticles. Soft Matter 9:4969–4976. https://doi.org/10.1039/c3sm27578g Popescu I, Suflet DM, Pelin IM, Chiţanu GC (2011) Biomedical applications of maleic anhydride copolymers. Rev Roum Chim 56:173–188 Regelson W (1979) The biologic activity of polyanions: Past history and new prospectives. J Polym Science: Polym Symposia 66:483–538. https://doi.org/10.1002/polc.5070660144 Breslow DS (1976) Biologically Active Synthetic Polymers. Pure Appl Chem 46:103–113. https://doi.org/10.1351/pac197646020103 Hainsworth JD, Forbes JT, Grosh WW, Greco FA (1986) Phase I study of MVE-2 evaluating toxicity and biologic response modification capability. Cancer Immunol Immunotherapy 22:68–71. https://doi.org/10.1007/BF00205719 Rinehart J, LaForge J, Gochnour D, Neidhart J (1987) Phase II trial of MVE-II in metastic malignant melanoma. Cancer Immunol Immunotherapy 24:244–246. https://doi.org/10.1007/BF00205637 Butler GB, Polym Sci J (2000) Polym Chem 38:3451–3461. https://doi.org/10.1002/1099-0518(20001001)38:193.0.CO;2-N Kunitake T, Tsukino M (1979) Radical cyclocopolymerization of divinyl ether and maleic anhydride. A 13C-NMR study of the polymer structure. J Polym Sci Polym Chem Ed 17:877–888. https://doi.org/10.1002/pol.1979.170170325 Gorshkova MY, Lebedeva TL, Stotskaya LL, Slonim IY (1996) Study of the structure of divinyl ether-maleic anhydride copolymer by spectroscopic methods. Polym Sci Ser A 38:1094–1096 Volkova IF, Gorshkova MY, Ivanov PE, Stotskaya LL (2002) New scope for synthesis of divinyl ether and maleic anhydride copolymer with narrow molecular mass distribution. Polym Adv Technol 13:1067–1070. https://doi.org/10.1002/pat.234 Malinovskaya Y, Melnikov P, Baklaushev V et al (2017) Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells. Int J Pharm 524:77–90. https://doi.org/10.1016/J.IJPHARM.2017.03.049 Zhukova V, Osipova N, Semyonkin A et al (2021) Fluorescently labeled plga nanoparticles for visualization in vitro and in vivo: The importance of dye properties. Pharmaceutics 13:1145. https://doi.org/10.3390/pharmaceutics13081145 Maksimenko O, Malinovskaya J, Shipulo E et al (2019) Doxorubicin-loaded PLGA nanoparticles for the chemotherapy of glioblastoma: Towards the pharmaceutical development. Int J Pharm 572:118733. https://doi.org/10.1016/j.ijpharm.2019.118733 Operti MC, Bernhardt A, Grimm S et al (2021) PLGA-based nanomedicines manufacturing: Technologies overview and challenges in industrial scale-up. Int J Pharm 605:120807. https://doi.org/10.1016/j.ijpharm.2021.120807 Gorshkova MY, Volkova IF, Grigoryan ES, Valuev LI (2020) Sodium Alginate Interpolymer Complexes as a Platform for pH-Tunable Drug Carriers. Polym Sci - Ser B 62:678–684. https://doi.org/10.1134/S1560090420060044 Liu M, Zheng Y, Liu Y et al (2020) Effects of poly(vinyl alcohol) and poly(acrylic acid) on interfacial properties and stability of compound droplets. Int J Hydrogen Energy 45:2925–2935. https://doi.org/10.1016/j.ijhydene.2019.11.129 Jha PK, Desai PS, Li J, Larson RG (2014) pH and salt effects on the associative phase separation of oppositely charged polyelectrolytes. Polym (Basel) 6:1414–1436. https://doi.org/10.3390/polym6051414 Sanchez-Gaytan BL, Fay F, Hak S et al (2017) Real-Time Monitoring of Nanoparticle Formation by FRET Imaging. Angewandte Chemie - Int Ed 56:2969–2972. https://doi.org/10.1002/anie.201611288 Li Z, Kovshova T, Malinovskaya J et al (2023) Modeling the Drug delivery Lifecycle of PLG Nanoparticles Using Intravital Microscopy. https://doi.org/10.1002/smll.202306726 . Small 2306726 Gorshkova MY, Lebedeva TL, Stotskaya LL (1996) Type of bond in products of the interaction between primary amines and the copolymer of divinyl ether and maleic anhydride. Russ Chem Bull 45:1628–1634. https://doi.org/10.1007/BF01431799 Gruber S, Nickel A (2023) Toxic or not toxic? The specifications of the standard ISO 10993-5 are not explicit enough to yield comparable results in the cytotoxicity assessment of an identical medical device. Front Med Technol 5:1195529. https://doi.org/10.3389/fmedt.2023.1195529 Neun BW, Dobrovolskaia MA (2011) Method for In Vitro Analysis of Nanoparticle Thrombogenic Properties. Methods Mol Biol 697:225–235. https://doi.org/10.1007/978-1-60327-198-1_24 Fornaguera C, Solans C (2017) Methods for the in vitro characterization of nanomedicines—biological component interaction. J Pers Med 7:2. https://doi.org/10.3390/jpm7010002 Neun BW, Cedrone Edward, Dobrovolskaia MA (2020) NCL Method ITA-1: Analysis of Hemolytic Properties of Nanoparticles. Methods in Molecular Biology. https://doi.org/10.17917/V9AP-D094 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Mendeleev University of Chemical Technology of Russia","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Malinovskaya","suffix":""},{"id":319400682,"identity":"46d3b3b6-2f44-4d87-b9c1-616e4f8f770f","order_by":8,"name":"Tatyana Kovshova","email":"","orcid":"","institution":"D. I. Mendeleev University of Chemical Technology of Russia","correspondingAuthor":false,"prefix":"","firstName":"Tatyana","middleName":"","lastName":"Kovshova","suffix":""},{"id":319400684,"identity":"5efac922-1b12-4f80-ac80-56db263aacf3","order_by":9,"name":"Svetlana Gelperina","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYFAC5gYgIQHlVBClhRFZyxkgZiNOC4zdRoQWeffGtgcf91jk8TewP2D4OG9bYv/83gfMlW24tRieOdhuOOOZRLHEAR4DxpnbbifOOMZuwHgWn5YZiW3SPAckEhsO8DAw8wK1bGBjY2BsxKdl/sM26T9ALfMPsD9g/juHCC3yEoxt0gxALRsOMBgwMzYQocWAJ7HdsAeoZeNhHoODPcduG884lsZwsOEcHlvaDx978ONAXeK84+0PH/youS3b33yM8WFDGR5bDsDigZmB4QBM9AB2xVBbGoiI7VEwCkbBKBjhAAA61FP6pVpoeQAAAABJRU5ErkJggg==","orcid":"","institution":"D. I. Mendeleev University of Chemical Technology of Russia","correspondingAuthor":true,"prefix":"","firstName":"Svetlana","middleName":"","lastName":"Gelperina","suffix":""}],"badges":[],"createdAt":"2024-06-17 13:16:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4594368/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4594368/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59599543,"identity":"9ab22190-b834-4a8c-bc4b-c9224f32f139","added_by":"auto","created_at":"2024-07-03 16:32:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":192388,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of DIVEMA in its anhydride form with tetrahydropyran (\u003cem\u003ea\u003c/em\u003e) or tetrahydrofuran cycles (\u003cem\u003eb\u003c/em\u003e) and acidic (hydrolyzed) form with the tetrahydrofuran cycle (\u003cem\u003ec\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/3a77f7bfaa51f49bb2df61a1.png"},{"id":59599544,"identity":"92faf046-2a87-4620-a9d0-037bd2a1ef62","added_by":"auto","created_at":"2024-07-03 16:32:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30985750,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrographs of nanoparticles obtained by homogenization: (\u003cem\u003ea\u003c/em\u003e) DIVEMA/PLGA NP (DIVEMA MM 20 kDa; DIVEMA/PLGA mass ratio = 1:8); (\u003cem\u003eb\u003c/em\u003e) PLGA NP (scanning electron microscope JSM 6510 LV series, Jeol Ltd., Japan; samples sputtered with platinum)\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/846264d2cafe3854aa1a799a.png"},{"id":59599540,"identity":"907c3699-6dcc-49de-bd3d-24e5d53439ac","added_by":"auto","created_at":"2024-07-03 16:32:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49744,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared (FTIR) spectra of DIVEMA/PLGA NP (\u003cem\u003e1\u003c/em\u003e), DIVEMA (\u003cem\u003e2\u003c/em\u003e) and PLGA NP (\u003cem\u003e3\u003c/em\u003e) (DIVEMA/PLGA mass ratio = 1:1.2, DIVEMA MM 20 kDa)\u003c/p\u003e","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/af4b86bfc183338445348d64.png"},{"id":59599547,"identity":"0367e26d-9f65-4031-ac51-1fcdb7e544b8","added_by":"auto","created_at":"2024-07-03 16:32:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45847,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential Fourier transform infrared (FTIR) spectra: (\u003cem\u003e1\u003c/em\u003e) PLGA NP, (\u003cem\u003e2\u003c/em\u003e) DIVEMA/PLGA NP, (\u003cem\u003e3\u003c/em\u003e) differential spectrum (DIVEMA/PLGA mass ratio = 1:1.2, DIVEMA MM 20 kDa)\u003c/p\u003e","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/280eee05f12bd1613178646c.png"},{"id":59599545,"identity":"6b32f3b5-2ada-4c45-894a-c8fafa4dd39f","added_by":"auto","created_at":"2024-07-03 16:32:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":382324,"visible":true,"origin":"","legend":"\u003cp\u003eAverage hydrodynamic diameters of DIVEMA/PLGA NP and PLGA NP measured at different pH and ionic strength of the medium. A ‑ nanoparticles prepared by nanoprecipitation (DIVEMA/PLGA ratio = 1:1.2, DIVEMA MM 20 kDa); B ‑ nanoparticles prepared by the homogenization – solvent evaporation technique (DIVEMA/PLGA ratio = 1:8, DIVEMA MM 20 kDa)\u003c/p\u003e","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/5648112e478dadf2ff4e2b48.png"},{"id":59599542,"identity":"48a355a1-75ec-4785-ae5c-fdac4a99e99a","added_by":"auto","created_at":"2024-07-03 16:32:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2217485,"visible":true,"origin":"","legend":"\u003cp\u003eGel permeation chromatography analysis of fluorescently labeled polymers (UV detection): \u003cem\u003e(a)\u003c/em\u003eDIVEMA-Cy3 (solid) and DIVEMA (dash); \u003cem\u003e(b)\u003c/em\u003e PLGA-Cy5 (solid) and PLGA (dash)\u003c/p\u003e","description":"","filename":"Onlinefigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/ed3c441afdd7d7baad8fdf3e.png"},{"id":59599541,"identity":"fd19641c-0cf5-44a1-b123-7eae1ba22907","added_by":"auto","created_at":"2024-07-03 16:32:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":290948,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of DIVEMA-Cy3/PLGA NP (DIVEMA-Cy3:PLGA ratio = 1:2.6) and DIVEMA-Cy3/PLGA-Cy5 NP (DIVEMA-Cy3:PLGA-Cy5 ratio = 1:2.6) registered at excitation wavelengths of Cy3 and Cy5 labels (530 nm and 630 nm, respectively). (\u003cem\u003e1\u003c/em\u003e) DIVEMA-Cy3/PLGA NP (λ\u003csub\u003eex\u003c/sub\u003e 530 nm); (\u003cem\u003e2\u003c/em\u003e) DIVEMA-Cy3/PLGA-Cy5 NP (λ\u003csub\u003eex\u003c/sub\u003e 530 nm); (\u003cem\u003e3\u003c/em\u003e) DIVEMA-Cy3/PLGA-Cy5 NP (λ\u003csub\u003eex\u003c/sub\u003e 630 nm). Cy3 concentration in both nanosuspensions ~1.27 x 10\u003csup\u003e-7 \u003c/sup\u003eM\u003c/p\u003e","description":"","filename":"Onlinefigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/9ea01c1f629049817db33927.png"},{"id":59599546,"identity":"aa12afd1-7be3-46e7-be9e-4767633dc650","added_by":"auto","created_at":"2024-07-03 16:32:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":237576,"visible":true,"origin":"","legend":"\u003cp\u003eProfiles of doxorubicin release from PLGA-Dox NP (a) and DIVEMA/PLGA-Dox NP (b) (0.9% NaCl, 37˚C). All nanoparticles were prepared by the double emulsion technique (w-o-w); the DIVEMA:PLGA:Dox mass ratios were 0:20:1 and 2:20:1 for PLGA-Dox NP and DIVEMA/PLGA-Dox NP, respectively.\u003c/p\u003e","description":"","filename":"Onlinefigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/3a01c04b1c4d3f98e2fc3514.png"},{"id":71774759,"identity":"72d1cb9a-73c9-4f6d-8972-21e6925c2bc0","added_by":"auto","created_at":"2024-12-18 13:02:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6592852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594368/v1/2cf0a52b-a40d-4ab1-8aa4-a51b4412ef88.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hybrid DIVEMA/PLGA nanoparticles as the potential drug delivery system","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe nanoparticles made of biodegradable and biocompatible copolymers of lactic and glycolic acids (PLGA) are widely used as nanocarriers for drug delivery [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, similarly to other colloidal nanocarriers, the intravenously injected PLGA nanoparticles (PLGA NP) tend to accumulate in the organs of the mononuclear phagocyte system (MPS) that is specialized in the clearance of both endogenous and exogenous particulates from the blood. This biodistribution pattern is beneficial for drug delivery to macrophage-rich organs (such as liver or spleen) but interferes with delivery to other targets [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, the scarcity of the reactive groups on the surface of the PLGA NP, limited to the end carboxylic groups of PLGA, hampers attachment of bioligands that could enable receptor-mediated transport of such delivery systems to the targets outside the MPS. The commonly used solution of this problem is the employment of the nanoparticles composed of PEG-PLGA copolymers that have demonstrated effective avoidance of macrophages whereas the terminal groups of PEG moieties could be used for conjugation with the targeting bioligand. However, certain disadvantages of this technology, such as, for example, PEG immunogenicity inspired the search for alternative approaches [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, one of the approaches involves modification of PLGA nanoparticles with polyelectrolytes, which, on one hand, increases hydrophilicity of their surface and on the other hand provides numerous functional groups for vectorization. Indeed, a number of studies are focused on fabrication of the PLGA nanoparticles modified with cationic polymers, which helps to improve their loading with nucleic acids (i.e. plasmid DNA) and proteins (i.e. antigens) by electrostatic interactions [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The drawback of the positively charged nanoparticles is that they are potentially more cytotoxic than negative or neutral ones, which is attributed to their stronger interaction with negatively charged cellular membranes that may cause the membrane damage [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother option is modification of the nanoparticles with biocompatible polyanions, such as for example copolymers of maleic anhydride that proved to be suitable for many biomedical applications [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An attractive candidate for this approach is the copolymer of divinyl ether with maleic anhydride widely known as DIVEMA. This remarkable polymer extensively studied in the 70s and 80s exhibits the broad spectrum of biological activities such as induction of biological response to tumors, antiviral and antibacterial activity explained most probably by induction of interferon production and/or macrophage activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to this \u003cem\u003eper se\u003c/em\u003e activity, DIVEMA became one of the first synthetic polymers clinically tested as the anticancer agent; however, despite the antitumor activity evidenced in numerous preclinical studies, no significant effect was observed in humans [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. DIVEMA was synthesized in 1951 by G.B. Butler by radical copolymerization of maleic anhydride with divinyl ether yielding the copolymer with alternating structure (mole ratio of divinyl ether: maleic anhydride is 1:2) that contains the cycle developed from both monomers. This reaction was recognized as a new type of polymerization designated as \u0026ldquo;cyclocopolymerization\u0026rdquo; [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Initially, Butler assumed that the cycle had the tetrahydropyran structure, four atoms of which were contributed by the diene and two atoms by the maleic anhydride (that is why the copolymer became known also as Pyran copolymer) (Fig.\u0026nbsp;1). Further studies using \u003csup\u003e13\u003c/sup\u003eC-NMR suggested that the polymer chain could contain both tetrahydropyran and tetrahydrofuran cycles (Fig.\u0026nbsp;1) [12]. The results of Kunitake et al. indicated that the cycle structure could depend on the solvent polarity where the non-polar solvent favored the formation of six-membered cycles [16]. However later, Gorshkova et al. demonstrated that synthesis of DIVEMA in both chloroform (non-polar) and acetone (polar) led to formation of the polymer containing predominantly tetrahydrofuran cycles [17].\u003c/p\u003e \u003cp\u003eWhile the role of the cycle structure in the biological properties of DIVEMA has not been elucidated, both its activity and toxicity were shown to be dependent on its molecular mass (MM) and molecular mass distribution (MMD). Thus, the results of the preclinical and clinical studies exhibited the acceptable toxicity of the polymer with the MM of 15\u0026ndash;20 kDa and narrow MMD (tolerable single intravenous dose in humans 2500 mg), whereas higher MM and wide MMD were associated with the increase of toxicity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough DIVEMA failed as the antitumor agent or immunomodulator, it may still be valuable for the purpose of drug delivery. Indeed, apart from its versatile biological activity, the advantages of DIVEMA, as the potential constituent of the nanoparticle-based delivery system, are its relatively low toxicity, facile synthesis, and abundant carboxylic groups (four carboxylic groups per each monomeric unit, Fig.\u0026nbsp;1) suitable for the surface modification.\u003c/p\u003e \u003cp\u003eTherefore, the objective of the present study was to investigate the possibility to prepare the hybrid DIVEMA/PLGA nanoparticles and evaluate its potential as the drug delivery system. Considering the aforementioned toxicological data, the experiments were performed using the DIVEMA sample with MM of 20 kDa synthesized for this study. The sample with MM of 80 kDa was used in some experiments for comparison.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003ePLGA (RESOMER\u0026reg; RG 502H, lactide/glycolide\u0026thinsp;=\u0026thinsp;50:50 mol/mol; acid terminated; MM 7,000\u0026ndash;17,000 Da; η\u0026thinsp;=\u0026thinsp;0.16\u0026ndash;0.24 dL/g) was purchased from Evonik R\u0026ouml;hm GmbH (Germany). N-(3-dimethylaminopropyl)-N\u0026prime;-ethylcarbodiimide hydrochloride (EDC), N,N\u0026prime;-dicyclohexylcarbodiimide (DCC), N\u0026ndash;hydroxysuccinimide (NHS), diisopropylethylamine (DIPEA), bovine serum albumin (BSA), polyvinyl alcohol (PVA, 9\u0026ndash;10 kDa, 80% hydrolyzed), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (USA). 2-Iminothiolane hydrochloride (Traut's reagent) was purchased from ThermoFisher Scientific (USA). Maleimide PEG3500 Amine (MAL-PEG3500-NH\u003csub\u003e2\u003c/sub\u003e, TFA salt, MM 3500 Da) was from JenKem Technology (USA). The reactive derivatives of the fluorescent dyes cyanine5 (Cy5) amine and cyanine3 (Cy3) amine were purchased from Lumiprobe (Russia). All other chemicals were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis and analysis of divinyl ether and maleic anhydride copolymer (DIVEMA)\u003c/h2\u003e \u003cp\u003eThe DIVEMA copolymer with an alternating (1:2) structure was synthesized by radical copolymerization of divinyl ether and maleic anhydride as described previously in [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. All reagents were thoroughly purified before the reaction: maleic anhydride was sublimated twice; divinyl ether was distilled over metallic Na; 2,2\u0026rsquo;-azobisisobutyronitrile (AIBN) used as an initiator was recrystallized twice from methanol and then dried in vacuum. The reaction was performed in the sealed glass ampoules in dry acetone that was purified and distilled immediately before use. Maleic anhydride and divinyl ether were added at the molar ratio of 2:1; the total monomer concentration was 1.28 mol/L; the AIBN concentration was 1.7 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e mol/L. The polymerization was performed at 60 \u0026deg;C. Tetrahydrofuran (THF, 22% v/v or 7% v/v) was added in the reaction mixture as the chain transfer agent. The resulting polymer was purified from low-molecular impurities by extraction in ether and then dried to a constant mass in vacuum.\u003c/p\u003e \u003cp\u003eThe molecular mass (MM) and mass distribution (MMD) of DIVEMA and DIVEMA-Cy3 conjugates were analyzed by gel permeation chromatography as described in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] using the Gilson liquid chromatography system (Gilson Inc., USA) equipped with the refractive index and UV detectors and TSK-gel GMPW column (7.5 mm i.d. x 600 mm) with TSK guard PWH column (Tosoh Corp., Japan). Borate buffer (pH 10.2) with addition of 0.2M NaCl was used as the mobile phase; the elution rate was 1 mL/min. A series of poly(ethylene glycol)-poly(ethylene oxide) (PEG-PEO) standards with narrow MMD (Polymer Standards Service GmbH, Germany) was used to obtain a calibration curve that was a straight line for molecular masses in the range of 1.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e\u0026ndash;1.2\u0026times;10\u003csup\u003e6\u003c/sup\u003e Da. The intrinsic viscosity was measured at 25 \u0026deg;C in the same borate buffer using the Ubbelohde suspended-level type capillary viscometer (Canon, England).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of DIVEMA/PLGA nanoparticles\u003c/h2\u003e \u003cp\u003eThe nanoparticles were prepared by the homogenization \u0026ndash; solvent evaporation technique or by nanoprecipitation. In the homogenization \u0026ndash; solvent evaporation technique, DIVEMA and PLGA were used in the mass ratio of ~\u0026thinsp;1:8. The solutions of PLGA (600 mg, in 7.2 mL of dichloromethane [DCM]) and DIVEMA (72 mg in 4.8 mL of acetone) were mixed and this organic phase was added to 60 mL of a 0.5% aqueous solution of polyvinyl alcohol (PVA, 9\u0026ndash;10 kDa) used as surfactant. The mixture was first emulsified using the high-shear homogenizer (Ultra-Turrax T18, IKA, Germany; 23600 rpm), and then this coarse emulsion was further processed using the high-pressure homogenizer (Microfluidics M-110P, Microfluidics, USA; 15000 psi). Then the organic solvent was removed under vacuum; the resulting suspension was separated from residual DIVEMA by centrifugation and thorough washing with water and then freeze-dried with 2.5% (w/v) of D-mannitol as cryoprotectant. The plain PLGA nanoparticles were obtained in a similar way; in this case, 4.8 mL of acetone without DIVEMA was added to the polymer solution in DCM.\u003c/p\u003e \u003cp\u003eIn the case of nanoprecipitation, the solution of PLGA (60 mg) and DIVEMA (50 mg, 23 mg, or 7.5 mg for DIVEMA/PLGA mass ratios of 1:1.2, 1:2.6, 1:8, respectively) in 6 mL of the ACN - acetone mixture (1:2 v/v) was added dropwise to 60 mL of a 0.5% aqueous PVA solution under stirring and incubated for 3 h. The resulting nanosuspension was processed and freeze-dried as described above. The plain PLGA nanoparticles were obtained similarly without addition of DIVEMA.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4. Preparation of fluorescently labeled nanoparticles for fluorescence spectroscopy study\u003c/b\u003e DIVEMA (MM 20 kDa) was labeled with the Cyanine3 dye (Cy3, λ\u003csub\u003eex\u003c/sub\u003e 555 nm, λ\u003csub\u003eem\u003c/sub\u003e 570 nm) by covalent binding of its water-soluble amine derivative (Cy3 amine) to carboxylic groups via the NHS/DCC coupling reaction. The solution of N-hydroxysuccinimide (NHS, 23 mg in 1 mL of acetone) was added to the DIVEMA solution (600 mg in 30 mL of acetone), and the mixture was stirred for 30 min in the dark. Then N,N'-dicyclohexylcarbodiimide (DCC, 14 mg in 0.5 mL of acetone) and Cy3 amine (18 mg in 1.5 mL of acetone-EtOH mixture) were added, and the mixture was stirred for another 3 h. Then the polymer was precipitated into the 10-fold volume of ether; the precipitate was separated by filtration, washed with chloroform and dried in vacuum. The polymer precipitation procedure was repeated three times. The absence of the unbound dye was monitored by the absence of absorption at 543 nm in the UV spectra of the washing solvent. The dye content in the DIVEMA-Cy3 conjugate was determined by UV spectroscopy (λ\u003csub\u003emax\u003c/sub\u003e 552 nm). The conjugate containing 2.7% w/w of the dye (dye-to-polymer ratio of 1:40, w/w) was used in the experiments.\u003c/p\u003e \u003cp\u003eThe DIVEMA-Cy3 conjugate was also analyzed by gel permeation chromatography as described above using the refractive index detector and the UV detector.\u003c/p\u003e \u003cp\u003ePLGA was labeled with the Cyanine5 dye (Cy5, λ\u003csub\u003eex\u003c/sub\u003e 651 nm, λ\u003csub\u003eem\u003c/sub\u003e 670 nm) by covalent binding of its water-soluble derivative Cy5 amine to the terminal carboxylic groups of PLGA via the NHS/EDC coupling reaction as described earlier [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, solutions of Cy5 amine, DIPEA, EDC, and NHS in DCM were added to the PLGA solution in DCM. The reaction mixture was incubated for 48 h under continuous stirring at room temperature in the dark. The obtained solution was washed 3 times with water and water/methanol (1:1) mixture. The organic phase was separated and dried over anhydrous sodium sulfate with subsequent evaporation. The obtained sediment was dissolved in ethyl acetate, and then added to the tenfold volume of hexane to precipitate the polymer. The precipitate was dried in vacuum. The content of Cy5 in the conjugate was measured spectrophotometrically. The PLGA-Cy5 conjugate with a dye-to-polymer ratio of 1: 600 (w/w) was used in the experiments.\u003c/p\u003e \u003cp\u003eThe PLGA-Cy5 conjugate was analysed by gel permeation chromatography using the Waters HPLC system equipped with a set of Styrogel HR5E and HR4E columns (300 mm \u0026times; 7.8 mm). Tetrahydrofuran was used as a solvent and eluent at the flow rate of 1.00 mL/min. Polymer solutions were prepared at the known concentration (ca. 1 mg/mL), injection volume was 50 \u0026micro;l. Data were collected using the refractive index detector (Waters 2414 RI Detector) and the UV detector (Milton Roy UV-detector 3100 model, λ 264 nm). Data analysis was performed with the Z-lab software. The system was calibrated using the polystyrene standards set. Formation of the PLGA-Cy5 conjugate was also confirmed by the TLC method on silica gel-coated plates using the DCM/methanol/water (6.5:2.5:0.4 v/v) mixture as eluent.\u003c/p\u003e \u003cp\u003eThe dual-labeled DIVEMA-Cy3/PLGA-Cy5 nanoparticles were prepared by nanoprecipitation as described above using the DIVEMA/PLGA mass ratio of 1:2.6. The 1:1 mixture of the PLGA-Cy5 conjugate and non-modified PLGA was used as the PLGA constituent.\u003c/p\u003e \u003cp\u003eThe fluorescence spectra of the labeled nanoparticles were registered using the RF-6000 spectrofluorometer (Shimadzu, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Physicochemical characterization of nanoparticles\u003c/h2\u003e \u003cp\u003eThe average hydrodynamic diameter (ZaveD) and polydispersity index (PDI) of the nanoparticles were determined by the dynamic light scattering method (DLS) using the Zetasizer Nano ZS instrument (helium-neon laser source with a wavelength of 632.8 nm, light scattering angle 173\u0026deg;, temperature 25\u0026deg;C; Malvern Instruments, UK, Great Britain). For measurements, the freeze-dried samples were resuspended in water and then the suspensions were diluted 50-fold with water. The zeta potential was measured by electrophoretic light scattering using the same instrument. Each measurement was repeated 4 times.\u003c/p\u003e \u003cp\u003eThe content of carboxylic groups on the nanoparticles or in the supernatants after nanoparticle separation by centrifugation was determined by potentiometric titration using the pH-meter with combined microelectrode.\u003c/p\u003e \u003cp\u003eThe Fourier transform infrared spectroscopy (FTIR) study was performed using the PerkinElmer Spectrum One FTIR spectrometer equipped with the universal ATR Sampling Accessories with Diamond/ZnSe crystal (PerkinElmer Inc., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Analysis of nanoparticle morphology by microscopy\u003c/h2\u003e \u003cp\u003eThe scanning electron microscopy (SEM) study of the nanoparticles was carried out using the JSM 6510 LV scanning electron microscope (Jeol Ltd., Japan) in the secondary electron mode. To improve the image quality the nanoparticles were washed from mannitol by repeated centrifugation. For this, the freeze-dried samples were reconstituted to the initial volume with distilled water, and then the suspension was subjected to ultrasonication and centrifuged at 20000 rpm for 30 min. This procedure was repeated 3 times using the same volume of water. Then the drop of the suspension was placed on the substrate, dried in air, sputtered with platinum for 30 s, and placed in the microscope. The DIVEMA/PLGA nanoparticles used for the SEM study were prepared by the homogenization \u0026ndash; solvent evaporation technique using DIVEMA with MM of 20 kDa, the PLGA : DIVEMA mass ratio was 8:1. The similarly prepared plain PLGA NP were used for comparison.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Synthesis of FITC-labeled bovine serum albumin (FITC-BSA)\u003c/h2\u003e \u003cp\u003eThe fluorescein isothiocyanate (FITC) solution in DMSO (0.0026 M, 1 mL) was added dropwise to the bovine serum albumin (BSA) solution in freshly prepared 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e/NaOH buffer (100 mg, 10 mL, pH 9). The BSA-FITC molar ratio was 1:2. The mixture was incubated under stirring at room temperature for 1 h and then for 15 h at 5\u0026ndash;6\u0026deg;C in the dark. Then the ammonium chloride was added into this solution to the concentration of 0.05 M, and stirring was continued for 1 h. The labeled protein was separated from free dye (FITC) by gel permeation chromatography using a Sephadex G-25 column (eluent \u0026ndash; 0.1 M phosphate buffer, pH 7.4). The purification procedure was repeated twice. The resulting FITC-BSA solution was freeze-dried without addition of a cryoprotectant. The FITC-BSA content and the FITC/protein (F/P) ratio were determined by spectrophotometry. To determine the content of FITC-BSA and the F/P ratio the FITC-BSA sample was dissolved in water and diluted 5-fold. The absorbance was measured at 494 nm and 280 nm. The protein concentration was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{P}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n} \\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\left(\\text{M}\\right)= \\frac{{(\\text{A}}_{280}-{\\text{A}}_{494})\\times \\text{C}\\text{F}(\\text{F}\\text{I}\\text{T}\\text{C})}{{\\epsilon }\\left(\\text{p}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n}\\right)} \\times \\text{d}\\text{i}\\text{l}\\text{u}\\text{t}\\text{i}\\text{o}\\text{n}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{c}\\left(\\text{B}\\text{S}\\text{A}\\right) \\left(\\text{M}\\right)= \\frac{{(\\text{A}}_{280}-{\\text{A}}_{494})\\times 0.3}{43824} \\times \\text{d}\\text{i}\\text{l}\\text{u}\\text{t}\\text{i}\\text{o}\\text{n}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\text{F}/\\text{P}= \\frac{{\\text{A}}_{494}}{68000 \\times \\text{C}\\left(\\text{B}\\text{S}\\text{A}\\right)}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe FITC-BSA concentration in this sample was 0.83 mg/mg lyophilisate, F/P\u0026thinsp;=\u0026thinsp;1.08.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Conjugation of DIVEMA/PLGA NP with FITC-BSA via PEG linker\u003c/h2\u003e \u003cp\u003e \u003cem\u003eThiolation of FITC-BSA using Traut\u0026rsquo;s reagent\u003c/em\u003e. The FITC-BSA solution (30 mg in 1.0 mL of PBS buffer, pH 8.0) was mixed with the Traut\u0026rsquo;s reagent solution (200 \u0026micro;g in 0.1 mL MilliQ water). The mixture was incubated for 1 h at 20\u0026deg;С under stirring (500 rpm). Then the unbound reagent was removed by filtration through the Microcon 30 kDa filter. The thiolated protein (FITC-BSA-SH) was separated from the unbound dye and other low molecular impurities by repeated ultrafiltration using the Microcon 30 kDa membrane filter (centrifugation at 20 000 rpm, 5\u0026deg;C, 20\u0026ndash;30 min), and then the protein solution was diluted to 2.0 mL with MQ water and freeze-dried.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePEGylation of FITC-BSA-SH using maleimide-PEG3500-amine\u003c/em\u003e. The FITC-BSA-SH solution (10 mg in 0.5 mL PBS, pH 7.4) was mixed with the MAL-PEG3500-NH\u003csub\u003e2\u003c/sub\u003e solution (3.0 mg of in 0.5 mL PBS, pH 7.4). The mixture was incubated for 48 h at 4\u0026deg;С, 500 rpm. The FITC-BSA-PEG-NH\u003csub\u003e2\u003c/sub\u003e fraction was purified by repeated ultrafiltration using the Microcon 30 kDa membrane filter, then diluted with water to 2.0 mL and freeze-dried.\u003c/p\u003e \u003cp\u003e \u003cem\u003eActivation of DIVEMA/PLGA nanoparticles carboxylic groups\u003c/em\u003e using N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). 14.0 mg of the freeze-dried nanoparticles (7.5 \u0026micro;M of \u0026ndash;COOH groups) was resuspended in 0.3 mL of MQ water, then 0.72 mg (3.9 \u0026micro;M) of EDC in 0.1 mL of MQ water and 1.75 mg (15.2 \u0026micro;M) of NHS in 0,1 mL of MQ were added. The mixture was incubated for 3 h at 20\u0026deg;С under stirring.\u003c/p\u003e \u003cp\u003e \u003cem\u003eConjugation of DIVEMA/PLGA NP with FITC-BSA-PEG-NH\u003c/em\u003e \u003csub\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sub\u003e. The FITC-BSA-PEG-NH\u003csub\u003e2\u003c/sub\u003e solution (5.0 mg in 0.5 mL of MQ water) was added to the activated NP and incubated for 12 h at 20\u0026deg;С under stirring. The unbound FITC-BSA-PEG-NH\u003csub\u003e2\u003c/sub\u003e was removed by centrifugation (20 000 rpm, 5\u0026deg;C, 20\u0026ndash;30 min) and purified by repeated ultrafiltration using the Microcon 30 kDa filter as described above. The nanoparticles were resuspended in 2.0 mL of MQ water using the US-bath and Vortex and then freeze-dried with 1% (w/v) of D-mannitol as a cryoprotectant. Alternatively, the surface of the DIVEMA/PLGA NP was modified with FITC-BSA-PEG by adsorption.\u003c/p\u003e \u003cp\u003eConjugation of FITC-BSA-PEG-NH\u003csub\u003e2\u003c/sub\u003e with the similarly activated plain PLGA NP was performed as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Preparation of doxorubicin-loaded DIVEMA/PLGA nanoparticles and evaluation of doxorubicin release rate\u003c/h2\u003e \u003cp\u003eThe DIVEMA/PLGA nanoparticles loaded with doxorubicin (DIVEMA/PLGA-Dox NP) were obtained by the high pressure homogenization - solvent evaporation technique (w/o/w double emulsion method). PLGA (600 mg in 3.6 mL of DCM) and DIVEMA (60 mg in 2.4 mL of acetone) were mixed, and then this organic phase was added to the solution of doxorubicin in 0.001 N HCl (30 mg in 4.7 mL). The mixture was emulsified using the disperser (Ultra-Turrax T18, 23600 rpm). This primary emulsion was added to the 1% PVA solution in phosphate-buffered saline (30 mL). The mixture was first emulsified using the Ultra-Turrax and then using the high-pressure homogenizer as described above. Thereafter, the organic solvent was removed under vacuum and then the suspension was lyophilized with the addition of 5% (w/v) D-mannitol as a cryoprotectant. When preparing the plain PLGA NP loaded with doxorubicin (PLGA-Dox NP), the polymer was dissolved in 6 mL of DCM. The total content of doxorubicin was determined spectrophotometrically (λ\u003csub\u003emax\u003c/sub\u003e 480 nm) after dissolution of the freeze-dried nanoparticles in DMSO. The encapsulation efficiency of doxorubicin was determined after separation of the nanoparticles by ultrafiltration using Amicon\u0026reg; Ultra-0.5 filters (Millipore, MWCO 100 kDa) and subsequent spectrophotometric measurement of the free drug concentration in the filtrate.\u003c/p\u003e \u003cp\u003eThe doxorubicin release rate from the DIVEMA/PLGA-Dox NP and PLGA-Dox NP was evaluated using the 0.9% sodium chloride solution as a physiologically relevant medium. The freeze-dried nanoparticles were resuspended in 25 mL of 0.9% sodium chloride (total doxorubicin concentration 65\u0026ndash;68 \u0026micro;g/mL) and placed in the shaker-incubator (200 rpm, 37\u0026deg;C). The samples were taken after 0, 1, 2, 3, 4, 6, and 24 h of incubation, and then the nanoparticles were separated by centrifugation (48300 g, 18\u0026deg;C, 30 min, Avanti JXN-30, Beckman Coulter, USA). The doxorubicin concentration in the supernatants was determined spectrophotometrically (λ\u003csub\u003emax\u003c/sub\u003e 480 nm). For each sample, 3 parallel measurements were carried out.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Evaluation of DIVEMA/PLGA NP cytotoxicity\u003c/h2\u003e \u003cp\u003eThe porcine kidney cells (LLC-PK1) and human hepatocellular carcinoma cells (Hep G2) were used for testing of the nanoparticle cytotoxicity \u003cem\u003ein vitro\u003c/em\u003e. The cell lines were obtained from the American Type Culture Collection (ATCC, USA). The cell viability was assessed using the MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma, USA) reduction assay. The test was conducted according to the manufacturer\u0026rsquo;s protocol. Briefly, MTT was dissolved in PBS at 5 mg/mL (stock solution). The cells were cultured in a humid atmosphere (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% air) using DMEM (Gibko) media containing 10% FBS (Biowest, USA) with the addition of a mixture of antibiotics (100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin, Gibco, USA), GlutaMAX (2 mM, Gibco, USA). The cells were seeded in a 96-well plate (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well), cultured for 24 h, and then incubated with different amounts of the nanoparticles in the range of 0-100 \u0026micro;g/mL. After 24-h incubation with the samples, the MTT stock solution (10 \u0026micro;l per 100 \u0026micro;l medium) was added to all wells, and the cells were incubated for another 4 hours at 37\u0026deg; C. Dimethylsufloxide (DMSO; 200 \u0026micro;l) was added to each well after removal of the supernatant to dissolve the dark blue crystals. After shaking the plate for 10 min, the cell viability was assessed by measuring the absorbance at 570 nm using the microplate scanning spectrophotometer; all measurements were performed three times. The results are represented as the average of three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Evaluation of DIVEMA/PLGA NP hemocompatibility\u003c/h2\u003e \u003cp\u003eThe experiments involving manipulations with human whole blood or blood plasma were approved by the local ethics committee of the N.N. Burdenko National Medical Research Center of Neurosurgery (Moscow, Russia; approval No. 11/2020). The hemolytic effects of the nanoparticles and their influence on coagulation of human blood plasma were assessed as described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe hemolytic effect of the nanoparticles was analyzed by the \u003cem\u003ein vitro\u003c/em\u003e colorimetric assay. Briefly, the DIVEMA/PLGA NP were added to the blood samples (human blood obtained from healthy volunteers) in the concentration of 100 \u0026micro;g/mL and incubated for 3 hours at 37\u0026deg;C. To determine total hemoglobin (tHb), 1% Triton X-100 was added to a separate blood sample to induce hemolysis (positive control). After incubation, the undamaged human red blood cells (RBC) were separated by centrifugation. The supernatants were transferred into 96-well plates. Ferricyanide oxidizes oxyhemoglobin to methemoglobin, and cyanide converts methemoglobin to cyanmethemoglobin, a red-colored derivative of hemoglobin, that is detected by spectrophotometry (λ\u003csub\u003emax\u003c/sub\u003e 540 nm). The acute \u003cem\u003ein vitro\u003c/em\u003e hemolytic properties of the nanoparticles were evaluated as the percent of hemolysis calculated as the ratio of the optical density of the sample to the optical density of the control.\u003c/p\u003e \u003cp\u003eThe influence of the DIVEMA/PLGA NP and PLGA NP on coagulation of human blood plasma (i.e. blood clotting) was evaluated by measurement of the prothrombin time (PT) after plasma incubation with the nanoparticles. The nanoparticles were added to the blood samples in the concentrations of 1, 10, and 100 \u0026micro;g/mL. The platelet-poor plasma was isolated by centrifugation (1500 g, 10 min, 25\u0026deg;C). The blood samples were incubated with the nanoparticles for 30 min at 37\u0026deg;C under continuous stirring and placed into the coagulometer. Then the calcium thromboplastin was added to activate the clot formation followed by the measurement of coagulation time. Measurements were performed in triplicates.\u003c/p\u003e \u003cp\u003eThe nanoparticle effect on human peripheral blood platelet aggregation was evaluated by flow cytometry as described in [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This approach is based on the measurement of the platelet activation level as a percentage of P-selectin expression on platelet surface. In brief, the platelet-rich plasma (PRP) was obtained by centrifugation (1000 rpm, 10 min) and incubated with nanoparticles in the concentrations of 1-100 \u0026micro;g/mL. The aggregation inductor ADP (adenosine diphosphate) was added to the positive control samples. Platelets were then stained by fluorescently-labeled anti-CD36 antibodies (FITC-conjugated); the subpopulation of activated platelets was determined using anti-CD36 and PE-conjugated anti-CD62 antibodies (indicate the level of P-selectin, Biolegend, USA). The samples were analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eDIVEMA was synthesized by radical cyclopolymerization of divinyl ether and maleic anhydride in dry acetone with AIBN used as the initiator. As shown previously by Gorshkova et al., this process led to formation of the copolymer with the tetrahydrofuran cycles which was confirmed by \u003csup\u003e13\u003c/sup\u003eC-NMR (data not shown) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. As mentioned above, the acceptable toxicity of DIVEMA was observed at the relatively low molecular mass of 18\u0026ndash;20 kDa and narrow MMD. Therefore, to enable the MM control THF was added in the reaction mixture as the chain transfer agent. The molecular mass parameters of the polymers obtained at different THF concentrations were analyzed by GPC as described in [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The copolymers obtained in the presence of 22% v/v or 7% v/v of THF had the molecular masses of 20 kDa and 80 kDa, respectively. For comparison, in the absence of THF the copolymer MM reached 220 kDa (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMolecular mass characteristics of DIVEMA samples synthesized in the presence of THF\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTHF content,\u003c/p\u003e\n \u003cp\u003e% v/v\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eDIVEMA molecular mass characteristics, Da\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e[\u0026eta;],\u003c/p\u003e\n \u003cp\u003eg/dl\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMn\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMw/Mn\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e220000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe PLGA NP are usually prepared by the high-pressure homogenization \u0026ndash; solvent evaporation technique or by nanoprecipitation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. In the present study, the DIVEMA/PLGA NP were prepared by both techniques using the mixture of DIVEMA (MM 20 kDa or 80 kDa) and PLGA; polyvinyl alcohol (0.5% aqueous solution, PVA MM 9\u0026ndash;10 kDa) was used as surfactant in both cases. The physicochemical parameters of the DIVEMA/PLGA NP and plain PLGA NP obtained by both methods at different DIVEMA/PLGA ratios are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Both methods yielded the hybrid nanoparticles with similar hydrodynamic diameters of ~\u0026thinsp;200 nm; the results were fairly reproducible (RSD\u003csub\u003e%\u003c/sub\u003e \u0026lt;15%). The nanoparticles obtained by high pressure homogenization had higher polydispersity, which is probably due to a higher initial concentration of PLGA used in this method. The molecular mass of DIVEMA did not exert any influence on the size of nanoparticles produced by nanoprecipitation: the nanoparticles produced using DIVEMA with MM of 20 kDa and 80 kDa had similar sizes of ~\u0026thinsp;200 nm. However, as compared with the plain PLGA NP, the DIVEMA/PLGA NP exhibited considerably bigger average hydrodynamic diameters, which suggests the presence of the more pronounced hydration shell. Accordingly, the content of the \u0026ndash;COOH groups in the DIVEMA/PLGA NP was considerably higher as compared to the plain PLGA NP (0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u0026nbsp;\u003cem\u003evs\u003c/em\u003e 0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mmol/g PLGA; DIVEMA/PLGA\u0026thinsp;=\u0026thinsp;1:8) indicating the presence of abundant polyanionic groups on the surface of the hybrid nanoparticles.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical parameters of DIVEMA/PLGA NP (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMethod of preparation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDIVEMA/ PLGA ratio,\u003c/p\u003e\n \u003cp\u003emg/mg\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eNanoparticle size and size distribution (DLS)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eZeta potential,\u003c/p\u003e\n \u003cp\u003emV\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverage size,\u003c/p\u003e\n \u003cp\u003enm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolydispersity index (PDI)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eDIVEMA 20 kDa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHigh pressure homogenization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e243\u0026thinsp;\u0026plusmn;\u0026thinsp;26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.205\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e180\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.240\u0026thinsp;\u0026plusmn;\u0026thinsp;0.090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eNanoprecipitation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e204\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.113\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-33.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e194\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e221\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.106\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-36.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eDIVEMA 80 kDa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNanoprecipitation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e176\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-39.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.105\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-34.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe nanoparticle morphology was investigated by scanning electron microscopy (SEM). For better image quality, the nanoparticles were washed form mannitol and coated with platinum. According to the DLS measurements, the DIVEMA/PLGA NP used for the SEM study had the hydrodynamic diameter of 203 nm (PDI 0.20). The plain PLGA NP were slightly smaller (mean diameter 180 nm, PDI 0.16). As seen from Fig. 2, both hybrid and plain nanoparticles had the spherical shape. Precise comparison of the size measurements obtained by SEM and DLS is not possible due to the high polydispersity of the nanoparticles; however, it appears that in the SEM micrographs the majority of the hybrid nanoparticles had smaller diameters than the diameter measured by DLS (100\u0026ndash;150 nm \u003cem\u003evs\u003c/em\u003e 203 nm). At the same time, in the case of the plain PLGA NP both methods yielded very similar results. This phenomenon may be attributed to the loss of the more considerable hydration shell of the DIVEMA/PLGA NP that was lost during drying.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFTIR spectroscopy study.\u003c/em\u003e The presence of DIVEMA in the DIVEMA/PLGA NP was confirmed using the FTIR spectroscopy. The sample of the thoroughly washed and freeze-dried DIVEMA/PLGA NP with the high content of DIVEMA (DIVEMA/PLGA mass ratio\u0026thinsp;=\u0026thinsp;1:1.2, DIVEMA MM 20 kDa) was used in the FTIR study to achieve better interpretation of the spectra. The shift of the PLGA carbonyl bond from 1746.77 cm\u003csup\u003e-1\u003c/sup\u003e to a higher frequency and its broadening in the spectrum of the DIVEMA/PLGA NP suggests the contribution of the DIVEMA`s carbonyls into this bond (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) and is also due to the formation of H-bonds [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. This shift became more considerable with the increase of the DIVEMA content in the nanoparticles (data not shown). Moreover, the increase of the DIVEMA content led to the decrease of the PLGA carbonyl band intensity and the simultaneous increase in the anhydride band area and intensity (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Appearance of additional bonds corresponding to the anhydride cycle (1759.79 cm\u003csup\u003e-1\u003c/sup\u003e) and carbonyl groups of hydrolyzed DIVEMA (1713.08 cm\u003csup\u003e-1\u003c/sup\u003e) was also observed in the differential spectra of the DIVEMA/PLGA NP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eParameters of carbonyl and anhydride cycle bonds in the FTIR spectra of PLGA NP and DIVEMA/PLGA NP (DIVEMA/PLGA mass ratio\u0026thinsp;=\u0026thinsp;1:1.2, DIVEMA MM 20 kDa)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eNanoparticle type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eDIVEMA/ PLGA ratio,\u003c/p\u003e\n \u003cp\u003emg/mg\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eBand parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eD1750/ D1380 carbonyl band, normalized spectra\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAnhydride cycle band\u003c/p\u003e\n \u003cp\u003eat 1759 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e,\u003c/p\u003e\n \u003cp\u003edifferential spectrum\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBand intensity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBand area, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD1759 band intensity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA1759 band area, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePLGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e184.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA/ PLGA NP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e182.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA/ PLGA NP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1:1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e173.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eFurthermore, the presence of the DIVEMA carboxylic groups on the nanoparticle surface was also confirmed by the dependence of the nanoparticle size measured by DLS on the pH and ionic strengths of the media. Thus, in contrast to the plain PLGA NP that maintained their size in the pH range of 3 to 6, the average size (DLS) of the hybrid nanoparticles, obtained by both nanoprecipitation and homogenization significantly depended on pH decreasing from 221 nm and 256 nm at pH 6.0 to 125 nm and 178 nm, respectively, at pH 3.0 (Fig.\u0026nbsp;5). This phenomenon is most probably explained by the decreased dissociation of DIVEMA carboxylic groups at lower pH level. The pH-dependent size of the polyelectrolyte-coated nanoparticles was also observed by other authors [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similar decrease of the DIVEMA/PLGA NP\u0026rsquo;s size observed in the 0.9% NaCl solution is due to the known coiling of polyelectrolytes in the presence of salts [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFluorescence spectroscopy study.\u003c/em\u003e The hybrid structure of the DIVEMA/PLGA NP was confirmed explicitly by the fluorescence analysis of the hybrid nanoparticles composed from the polymers labeled with the fluorescent dyes: PLGA was labeled with the Cyanine5 dye (Cy5, \u0026lambda;\u003csub\u003eex\u003c/sub\u003e 646 nm, \u0026lambda;\u003csub\u003eem\u003c/sub\u003e 662 nm) and DIVEMA was labeled with the Cyanine3 dye (Cy3, \u0026lambda;\u003csub\u003eex\u003c/sub\u003e 555 nm, \u0026lambda;\u003csub\u003eem\u003c/sub\u003e 570 nm). The choice of these dyes is based on their known donor - acceptor properties. This dye pair exhibits the distance-dependent F\u0026ouml;rster resonance energy transfer (FRET) phenomenon that appears only when both dye molecules are localized in the close proximity [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe water-soluble amine derivatives of the fluorescent dyes were bound covalently to the carboxylic groups of the polymers using the NHS/carbodiimide reactions. The covalent attachment of the dyes to the polymers was confirmed by the GPC analysis as described above in [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The chromatograms of the labeled and non-labeled polymers obtained using the UV detector are shown in Fig. 6.\u003c/p\u003e\n\u003cp\u003eThe dual-labeled DIVEMA-Cy3/PLGA-Cy5 NP were prepared by nanoprecipitation using the PLGA-Cy5 conjugate (1:1 mixture of the conjugate and non-modified PLGA) and DIVEMA conjugate with the Cy3. The physicochemical parameters of the DIVEMA-Cy3/PLGA-Cy5 NP were similar to that of the non-labeled nanoparticles (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical parameters of fluorescently labeled nanoparticles obtained by nanoprecipitation (DIVEMA MM 20 kDa, DIVEMA/PLGA mass ratio\u0026thinsp;=\u0026thinsp;1:2.6)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNanoparticle type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eNanoparticle size and size distribution (DLS)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eZeta potential,\u003c/p\u003e\n \u003cp\u003emV\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverage size, nm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolydispersity index (PDI)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA-Cy3/PLGA-Cy5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e193\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.040\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA-Cy3/PLGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e194\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.079\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA/PLGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e194\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.081\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eComparison of the DIVEMA-Cy3/PLGA-Cy5 NP fluorescence spectra registered at \u0026lambda;\u003csub\u003eex\u003c/sub\u003e 530 nm and 630 nm and the DIVEMA-Cy3/PLGA NP spectrum registered at \u0026lambda;\u003csub\u003eex\u003c/sub\u003e 530 nm revealed the decrease of the fluorescence intensity of Cy3 (donor) in parallel with the significant increase in the fluorescence intensity of Cy5 (acceptor) for the dual-labeled nanoparticles at \u0026lambda;\u003csub\u003eex\u003c/sub\u003e 530 nm (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTogether with the known stable retention of the fluorescence labels, the FRET phenomenon observed for the dual-labeled nanoparticles is the strong argument in favor of the close proximity of two polymers and stability of the hybrid particles in aqueous media. Altogether this system stability suggests the interaction between DIVEMA and PLGA that is most probably explained by the formation of H-bonds between DIVEMA hydroxyl and carbonyl groups and PLGA ester groups.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEnhanced surface functionality\u003c/em\u003e. The enhanced functionality of the DIVEMA/PLGA NP was demonstrated by the effective conjugation of the FITC-labelled PEGylated bovine serum albumin (FITC-PEG-BSA) chosen as the model biovector. The COOH-enriched nanoparticle surface enabled conjugation of 550 \u0026micro;g of protein per 1 mg of the nanoparticles, which is \u0026gt;\u0026thinsp;100-fold higher than the content of 4 \u0026micro;g/mg NP achieved by the protein conjugation with the end carboxylic groups of PLGA in the plain PLGA NP (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The latter result correlates with the data of other authors. The adsorption of FITC-BSA on the DIVEMA/PLGA NP was considerably less effective (only 118 \u0026micro;g/mg NP).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical parameters of DIVEMA/PLGA NP modified with FITC-BSA-PEG (representative data)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethod of FITC-BSA-PEG attachment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNanoparticle type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFITC-BSA-PEG content,\u003c/p\u003e\n \u003cp\u003e\u0026micro;g/mg NP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverage NP size (DLS), nm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolydispersity index (PDI)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZeta-potential,\u003c/p\u003e\n \u003cp\u003emV\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConjugation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA/PLGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e278\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.189\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdsorption\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDIVEMA/PLGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e267\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-31.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConjugation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePLGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e~\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e180\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.150\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProlongation of the drug release rate\u003c/em\u003e. Another potentially useful feature of the DIVEMA/PLGA NP such as the possibility to optimize the drug encapsulation and release rate was demonstrated using the antitumor antibiotic doxorubicin as the model drug. Indeed, the presence of DIVEMA led to a very considerable increase of the doxorubicin encapsulation efficiency in the hybrid nanoparticles as compared to the plain PLGA NP (97.5% and 50.7% for the DIVEMA/PLGA NP and PLGA NP, respectively, Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, the presence of DIVEMA also prolonged the drug release rate from the nanoparticles: approximately 20% and 50% of the drug was released from the DIVEMA/PLGA NP during the first 4 h and 24 h of the experiment, while the PLGA NP released \u0026sim;60% and \u0026sim;90% of the drug during the same time periods (Fig.\u0026nbsp;9). The influence of DIVEMA on the doxorubicin loading and release rate is most probably enabled by the electrostatic interaction between its numerous carboxylic groups and the amine group of doxorubicin as described earlier [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical parameters of doxorubicin-loaded DIVEMA/PLGA-Dox and PLGA-Dox nanoparticles (representative data)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDIVEMA/PLGA/\u003c/p\u003e\n \u003cp\u003eDox,\u003c/p\u003e\n \u003cp\u003emg/mg\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal drug content, mg/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEncapsulation efficiency of doxorubicin,\u003c/p\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAverage NP size, nm\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePolydispersity index (PDI)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eZeta-potential,\u003c/p\u003e\n \u003cp\u003emV\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0: 20:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e133\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.214\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2: 20:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e203\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEvaluation of cytotoxicity and hemocompatibility\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity and hemocompatibility of the DIVEMA/PLGA NP were assessed using the samples containing DIVEMA with MM 20 кDa or 80 kDa obtained by the nanoprecipitation method (DIVEMA/PLGA ratio\u0026thinsp;=\u0026thinsp;1:8, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The plain PLGA NP were used as control.\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity was assessed \u003cem\u003ein vitro\u003c/em\u003e in the porcine kidney epithelial cells LLC-PK1 and Hep G2 human hepatocellular carcinoma cells by the МТТ-test. The reduction of viability of both cell types after incubation with the DIVEMA/PLGA NP or plain PLGA NP in the concentration of up to 100 \u0026micro;g/mL did not exceed 20%, which is generally accepted as the non-toxic effect [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe hematotoxicity was evaluated by the generally accepted tests, such as the level of hemolysis, blood coagulation test, and platelet activation test as described earlier [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Both types of nanoparticles did not produce any significant influence on blood components.\u003c/p\u003e\n\u003cp\u003eThus, the hemolysis index determined as the percentage of positive control (blood samples after incubation with Triton X100) was found to be less than 2% for all samples, which is classified as \u0026ldquo;non-hemolytic\u0026rdquo; according to the NCL nanoparticle characterization protocols established by the US National Cancer Institute [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe DIVEMA/PLGA NP as well as the plain PLGA NP used as control did not influence the blood coagulation time at concentrations of up to 100 \u0026micro;g/mL: the prothrombin time (PT) values remained within the normal physiological range (12\u0026ndash;15 s) for all nanoparticle types; no statistically significant difference was observed between the nanoparticles. Incubation of platelet-rich plasma with both nanoparticle types did not induce platelet activation in all tested concentrations up to 100 \u0026micro;g/mL. The percent of the CD62P-positive cells used as a platelet activation marker did not significantly differ from control values.\u003c/p\u003e\n\u003cp\u003eLow cytotoxicity and good hemocompatibility of the PLGA NP correlated with the previously reported results [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe hybrid DIVEMA/PLGA nanoparticles appear to be the stable nanosystem that exhibits important advantages as compared to the plain PLGA nanoparticles. Due to the enhanced surface functionality this nanosystem offers not only the improved binding of biovectors but also the optimized drug carrier properties which will be useful for many drugs capable of electrostatic interaction with the carboxylic groups of DIVEMA. Taken together with low cytotoxicity and good hemocompatibility of the hybrid nanoparticles, these properties suggest that they could be considered as a promising and versatile nanocarrier. This is the first study that describes the DIVEMA/PLGA NP and demonstrates their potential as the drug delivery system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was fulfilled with the financial support from the Ministry of Science and Higher Education of the Russian Federation (state assignment project FSSM-2022-0003).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.G. and S.G. conceptualized the study. L.V., N.O., A.N., J.K., E.K., Yu.E., J.M., T.K., conducted the experiments; J.K. prepared all figures; M.G., T.K. and S.G. performed the data analysis and wrote the manuscript text. All authors have read and agreed to the published version of\u0026nbsp;the\u0026nbsp;manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe study was carried out within the framework of the State Program of TIPS RAS. The authors are grateful to the D. I. Mendeleev Center for Collective Use of Scientific Equipment for performing the analytical tests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRezvantalab S, Drude NI, Moraveji MK et al (2018) PLGA-based nanoparticles in cancer treatment. Front Pharmacol 9:1260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2018.01260\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2018.01260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGolombek SK, May JN, Theek B et al (2018) Tumor targeting via EPR: Strategies to enhance patient responses. Adv Drug Deliv Rev 130:17\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.addr.2018.07.007\u003c/span\u003e\u003cspan address=\"10.1016/j.addr.2018.07.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchubert J, Chanana M (2018) Coating Matters: Review on Colloidal Stability of Nanoparticles with Biocompatible Coatings in Biological Media, Living Cells and Organisms. Curr Med Chem 25:4553\u0026ndash;4586. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/0929867325666180601101859\u003c/span\u003e\u003cspan address=\"10.2174/0929867325666180601101859\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoler Besumbes E, Fornaguera C, Monge M et al (2019) PLGA cationic nanoparticles, obtained from nano-emulsion templating, as potential DNA vaccines. Eur Polym J 120:109229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.eurpolymj.2019.109229\u003c/span\u003e\u003cspan address=\"10.1016/j.eurpolymj.2019.109229\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWusiman A, Gu P, Liu Z et al (2019) Cationic polymer modified PLGA nanoparticles encapsulating alhagi honey polysaccharides as a vaccine delivery system for ovalbumin to improve immune responses. Int J Nanomed 14:3221\u0026ndash;3234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IJN.S203072\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S203072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTracey SR, Smyth P, Herron UM et al (2023) Development of a cationic polyethyleneimine-poly(lactic-co-glycolic acid) nanoparticle system for enhanced intracellular delivery of biologics. RSC Adv 13:33721\u0026ndash;33735. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/d3ra06050k\u003c/span\u003e\u003cspan address=\"10.1039/d3ra06050k\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFr\u0026ouml;hlich E (2012) The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomed 7:5577\u0026ndash;5591. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IJN.S36111\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S36111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin HP, Akimoto J, Li YK, Ito Y (2020) Selective Control of Cell Activity with Hydrophilic Polymer-Covered Cationic Nanoparticles. Macromol Biosci 20:2000049. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mabi.202000049\u003c/span\u003e\u003cspan address=\"10.1002/mabi.202000049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Malmstadt N (2013) Deformation and poration of lipid bilayer membranes by cationic nanoparticles. Soft Matter 9:4969\u0026ndash;4976. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c3sm27578g\u003c/span\u003e\u003cspan address=\"10.1039/c3sm27578g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePopescu I, Suflet DM, Pelin IM, Chiţanu GC (2011) Biomedical applications of maleic anhydride copolymers. Rev Roum Chim 56:173\u0026ndash;188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegelson W (1979) The biologic activity of polyanions: Past history and new prospectives. J Polym Science: Polym Symposia 66:483\u0026ndash;538. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/polc.5070660144\u003c/span\u003e\u003cspan address=\"10.1002/polc.5070660144\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreslow DS (1976) Biologically Active Synthetic Polymers. Pure Appl Chem 46:103\u0026ndash;113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1351/pac197646020103\u003c/span\u003e\u003cspan address=\"10.1351/pac197646020103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHainsworth JD, Forbes JT, Grosh WW, Greco FA (1986) Phase I study of MVE-2 evaluating toxicity and biologic response modification capability. Cancer Immunol Immunotherapy 22:68\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00205719\u003c/span\u003e\u003cspan address=\"10.1007/BF00205719\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRinehart J, LaForge J, Gochnour D, Neidhart J (1987) Phase II trial of MVE-II in metastic malignant melanoma. Cancer Immunol Immunotherapy 24:244\u0026ndash;246. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00205637\u003c/span\u003e\u003cspan address=\"10.1007/BF00205637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButler GB, Polym Sci J (2000) Polym Chem 38:3451\u0026ndash;3461. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/1099-0518(20001001)38:19\u0026lt;3451::AID-POLA10\u0026gt;3.0.CO;2-N\u003c/span\u003e\u003cspan address=\"10.1002/1099-0518(20001001)38:19%3C3451::AID-POLA10%3E3.0.CO;2-N\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKunitake T, Tsukino M (1979) Radical cyclocopolymerization of divinyl ether and maleic anhydride. A 13C-NMR study of the polymer structure. J Polym Sci Polym Chem Ed 17:877\u0026ndash;888. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pol.1979.170170325\u003c/span\u003e\u003cspan address=\"10.1002/pol.1979.170170325\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorshkova MY, Lebedeva TL, Stotskaya LL, Slonim IY (1996) Study of the structure of divinyl ether-maleic anhydride copolymer by spectroscopic methods. Polym Sci Ser A 38:1094\u0026ndash;1096\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolkova IF, Gorshkova MY, Ivanov PE, Stotskaya LL (2002) New scope for synthesis of divinyl ether and maleic anhydride copolymer with narrow molecular mass distribution. Polym Adv Technol 13:1067\u0026ndash;1070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pat.234\u003c/span\u003e\u003cspan address=\"10.1002/pat.234\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalinovskaya Y, Melnikov P, Baklaushev V et al (2017) Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells. Int J Pharm 524:77\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.IJPHARM.2017.03.049\u003c/span\u003e\u003cspan address=\"10.1016/J.IJPHARM.2017.03.049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhukova V, Osipova N, Semyonkin A et al (2021) Fluorescently labeled plga nanoparticles for visualization in vitro and in vivo: The importance of dye properties. Pharmaceutics 13:1145. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pharmaceutics13081145\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics13081145\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaksimenko O, Malinovskaya J, Shipulo E et al (2019) Doxorubicin-loaded PLGA nanoparticles for the chemotherapy of glioblastoma: Towards the pharmaceutical development. Int J Pharm 572:118733. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijpharm.2019.118733\u003c/span\u003e\u003cspan address=\"10.1016/j.ijpharm.2019.118733\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOperti MC, Bernhardt A, Grimm S et al (2021) PLGA-based nanomedicines manufacturing: Technologies overview and challenges in industrial scale-up. Int J Pharm 605:120807. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijpharm.2021.120807\u003c/span\u003e\u003cspan address=\"10.1016/j.ijpharm.2021.120807\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorshkova MY, Volkova IF, Grigoryan ES, Valuev LI (2020) Sodium Alginate Interpolymer Complexes as a Platform for pH-Tunable Drug Carriers. Polym Sci - Ser B 62:678\u0026ndash;684. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1134/S1560090420060044\u003c/span\u003e\u003cspan address=\"10.1134/S1560090420060044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Zheng Y, Liu Y et al (2020) Effects of poly(vinyl alcohol) and poly(acrylic acid) on interfacial properties and stability of compound droplets. Int J Hydrogen Energy 45:2925\u0026ndash;2935. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijhydene.2019.11.129\u003c/span\u003e\u003cspan address=\"10.1016/j.ijhydene.2019.11.129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJha PK, Desai PS, Li J, Larson RG (2014) pH and salt effects on the associative phase separation of oppositely charged polyelectrolytes. Polym (Basel) 6:1414\u0026ndash;1436. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/polym6051414\u003c/span\u003e\u003cspan address=\"10.3390/polym6051414\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Gaytan BL, Fay F, Hak S et al (2017) Real-Time Monitoring of Nanoparticle Formation by FRET Imaging. Angewandte Chemie - Int Ed 56:2969\u0026ndash;2972. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/anie.201611288\u003c/span\u003e\u003cspan address=\"10.1002/anie.201611288\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Kovshova T, Malinovskaya J et al (2023) Modeling the Drug delivery Lifecycle of PLG Nanoparticles Using Intravital Microscopy. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/smll.202306726\u003c/span\u003e\u003cspan address=\"10.1002/smll.202306726\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Small 2306726\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorshkova MY, Lebedeva TL, Stotskaya LL (1996) Type of bond in products of the interaction between primary amines and the copolymer of divinyl ether and maleic anhydride. Russ Chem Bull 45:1628\u0026ndash;1634. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01431799\u003c/span\u003e\u003cspan address=\"10.1007/BF01431799\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGruber S, Nickel A (2023) Toxic or not toxic? The specifications of the standard ISO 10993-5 are not explicit enough to yield comparable results in the cytotoxicity assessment of an identical medical device. Front Med Technol 5:1195529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmedt.2023.1195529\u003c/span\u003e\u003cspan address=\"10.3389/fmedt.2023.1195529\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeun BW, Dobrovolskaia MA (2011) Method for In Vitro Analysis of Nanoparticle Thrombogenic Properties. Methods Mol Biol 697:225\u0026ndash;235. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-60327-198-1_24\u003c/span\u003e\u003cspan address=\"10.1007/978-1-60327-198-1_24\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFornaguera C, Solans C (2017) Methods for the in vitro characterization of nanomedicines\u0026mdash;biological component interaction. J Pers Med 7:2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jpm7010002\u003c/span\u003e\u003cspan address=\"10.3390/jpm7010002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeun BW, Cedrone Edward, Dobrovolskaia MA (2020) NCL Method ITA-1: Analysis of Hemolytic Properties of Nanoparticles. Methods in Molecular Biology. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.17917/V9AP-D094\u003c/span\u003e\u003cspan address=\"10.17917/V9AP-D094\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hybrid nanoparticles, Divinyl ether-co-maleic anhydride copolymer (DIVEMA), Doxorubicin, Drug delivery systems, PLGA, Polyanion","lastPublishedDoi":"10.21203/rs.3.rs-4594368/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4594368/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe hybrid nanoparticles (NP) consisting of poly(lactic-co-glycolic acid) (PLGA) and polyanionic copolymer of divinyl ether with maleic anhydride (DIVEMA) were prepared by the high pressure homogenization \u0026ndash; solvent evaporation technique or by nanoprecipitation and evaluated by physicochemical and spectroscopic methods. The nanoparticles formed by PLGA (MM 7\u0026ndash;17 kDa) and DIVEMA (MM 20 kDa or 80 kDa) at mass ratios from 1.2:1 to 8:1 had the hydrodynamic diameter of ~\u0026thinsp;200 nm, negative zeta potentials of -33 to -40 mV, and were stable upon freeze-drying. The presence of DIVEMA in the PLGA nanoparticles improved their properties as the drug carrier. Thus, loading of the model drug doxorubicin was increased 2-fold and its release time was considerably extended. The enhanced surface functionality of the hybrid nanoparticles was demonstrated by a\u0026thinsp;~\u0026thinsp;5-fold higher content of the surface-conjugated PEGylated bovine serum albumin as compared with the plain PLGA nanoparticles. The DIVEMA/PLGA NP exhibited low cytotoxicity and good hemocompatibility. This is the first study that describes the DIVEMA/PLGA NP and demonstrates their potential as the drug delivery system.\u003c/p\u003e","manuscriptTitle":"Hybrid DIVEMA/PLGA nanoparticles as the potential drug delivery system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 16:32:43","doi":"10.21203/rs.3.rs-4594368/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68c4657e-60ac-4948-838c-e02f75a90337","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-18T12:53:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 16:32:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4594368","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4594368","identity":"rs-4594368","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}