Spectroscopic analysis of human serum albumin nanoparticles with encapsulated phenothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine) – continuation studies

Spectroscopic analysis of human serum albumin nanoparticles with encapsulated phenothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine) – continuation studies | 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 Spectroscopic analysis of human serum albumin nanoparticles with encapsulated phenothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine) – continuation studies Karolina Kulig, Patrycja Sarkowicz, Małgorzata Jeleń, Beata Morak-Młodawska, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6749623/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Nanoparticles (NPs) provide a potential opportunity to reduce toxicity, optimize drug effects, and properly distribute drugs in the body and/or overcome multidrug resistance. Human serum albumin (HSA) is widely used as a drug carrier due to its biocompatibility and specific affinity to cancer cells. 6-Acetylaminobutyl-9-chloroquino[3,2-b]benzo[ 1 , 4 ]thiazine (QBT) is a tetracyclic, acetylaminobutyl phenothiazine derivative in which one of the benzene rings has been replaced by a quinoline. This compound has shown very promising in vitro and in vivo biological properties. The aim of this study was the spectroscopic analysis of QBT and the development of albumin nanoparticles (HSA-NPs) with encapsulated QBT (QBT-HSA-NPs). This study is a continuation of attempts to encapsulate phenothiazine derivatives in nanoparticles. To examine the spectroscopic properties of QBT, UV-Vis spectroscopy was applied. To investigate the properties of QBT to be encapsulated in HSA nanoparticles, the desolvation method was used. By using scanning electron microscopy (SEM), the size and shape of the nanoparticles were ascertained. The QBT release study was determined using the sampling and separation method and the mathematical drug release kinetics mechanism was estimated. Changes in the secondary structure of HSA were verified using circular dichroism (CD) spectropolarimetry. QBT has an ability to absorb radiation in the UV-Vis range. The encapsulation efficiency was 97.44 ± 0.11%, confirming that QBT can be encapsulated in HSA nanoparticles. SEM examination showed smooth nanoparticles of their size of 101.445 ± 9.907 nm for QBT-HSA-NPs and 92.680 ± 12.797 nm for HSA-NPs. QBT released according to the zero-order mechanism, via QBT diffusion and HSA swelling. The presence of QBT in nanoparticles partially protected the secondary structure of HSA. The observed changes in the structure of native HSA, influenced by the presence of QBT at the molecular level, may not have a strong influence on the side effects generated in the in vivo system. Despite reports on albumin nanoparticles and QBT, no one has published studies on QBT encapsulation in nanoparticles to date. nanoparticles albumin phenothiazine derivative spectroscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Nanomaterials are one of the most rapidly evolving research topics. It is an interdisciplinary science field, the understanding of which requires knowledge from fields such as biology, chemistry, electronics physics or engineering [ 1 ]. Nanomaterials are one of the most rapidly evolving research topics. It is an interdisciplinary science field, the understanding of which requires knowledge from fields such as biology, chemistry, electronics physics or engineering [ 1 ]. Numerous studies show nanoparticles (NPs) as effective tools in many areas of biological and other sciences. They are used as drug carriers in both therapeutics and diagnostics. Their physicochemical properties, ability to bind the drug and release the therapeutic substance and low toxicity determine the clinical application of NPs. They are characterised by sizes in the range of 1-100 nm and their properties depend on the size and surface functionalization. The growing interest in nanostructures is consolidated by their ability to be imaged utilizing microscopic methods, such as scanning tunneling microscopy (STM), scanning transmission electron microscopy (STEM), or tandem electron microscopy (TEM) [ 2 ]. NPs' fate in the body is significantly influenced by their physicochemical properties, which significantly alter absorption, pharmacokinetics, clearance, cellular uptake, and cytotoxicity [ 3 ]. The chemical structure of NPs allows for the classification of them into three groups: organic, inorganic, and carbon nanoparticles [ 3 ]. The polymer that is the building block of the NPs as a drug delivery system, must follow several requirements. Firstly, it must be biodegradable or completely eliminated from the body in a timeframe that allows repeated administration without the danger of accumulation in the body. It is imperative that neither the polymer nor the product of its degradation be immunogenic or toxic. In addition, it should allow the synthesis of nanoparticles with appropriate properties in relation to the drug delivery target for which the nanoparticles have been designed [ 4 ]. Polymer NPs can serve as carriers for biologically active particles adsorbed on the surface or encapsulated inside them. Both natural and synthetic polymers can be used for synthesis. Natural polymers are recommended for the synthesis of nanoparticles due to their biodegradability. Advantages of polymeric NPs include high drug encapsulation efficiency, higher intracellular uptake compared to other drug carriers, higher stability of active substances, biocompatibility, and biodegradability. On the other hand, disadvantages include high production costs, risk of non-biodegradability, toxicity of solvents used for synthesis or brittleness [ 5 ]. Human serum albumin (HSA) was first used in the delivery of paclitaxel in the medication Abraxane. Early in 2005, it was introduced to the medical community [ 5 ]. Its easy availability, biocompatibility and ability to accumulate in tissues with a high metabolism have determined that albumin has become the most widely used natural polymer for the synthesis of therapeutic nanoparticles. The Federal Food and Drug Administration (FDA) has approved HSA for this purpose. It is a particularly attractive carrier for anti-cancer and anti-inflammatory drugs. Tumor-affected tissue is characterized by increased expression of the glycoprotein – secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin, which results in the accumulation of albumin and thus increased efficacy of cytostatic drugs encapsulated in protein nanoparticles [ 6 ]. In addition, HSA is one of the natural antioxidants which may have a role in increasing the efficacy of the nanoparticle [ 7 ]. Phenothiazines are a group of compounds with a broad spectrum of biological activities, including the following antipsychotic, antihistamine, antitussive and antiemetic. Also documented for this group of compounds are anticancer, antimicrobial and, related, against multidrug resistance (MDR) [ 12 ]. 6-Acetylaminobutyl-9-chloroquino[3,2-b]benzo[ 1 , 4 ]thiazine (QBT) is a four-ring phenothiazine derivative obtained by replacing one benzene ring with a quinoline system and introducing an acetylaminobutyl substituent to the thiazine nitrogen atom. QBT potently inhibited phytohemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells (PBMC), tumor necrosis alpha production, and growth of tumor cell lines. QBT shows comparable activity to cis-platinum against cancer cell lines A-431, L-1210 and SW-948. QBT proved to be suppressive in the models of delayed type hypersensitivity to ovalbumin and carrageenan induced footpad tests in mice. This compound was also effective in the amelioration of contact sensitivity to oxazolone and experimentally induced psoriasis. In in vivo mouse studies, the efficacy of the compound QBT in alleviating dextran sulphate sodium (DSS)-induced ulcerative colitis has been demonstrated [ 9 – 11 ]. The potential drug substance used in this research – QBT, is shown in Fig. 1 . The aim of this novelty study was to perform spectroscopic analysis of QBT and its encapsulation into HSA nanoparticles. The research conducted presents the use of QBT as an encapsulated substance in albumin nanoparticles prepared using the desolvation method, release study and confirmation of the above results by spectroscopic techniques. Despite reports on albumin nanoparticles and QBT, no one has published studies on QBT encapsulation in nanoparticles to data. The work also continues our series of studies on the encapsulation of phenothiazine derivatives in albumin nanoparticles. 2. Materials and methods Dmethyl sulfoxide (DMSO) and human serum albumin (HSA) with a minimum purity of 96% were acquired from Sigma Aldrich in Steinheim, Germany. Ethanol was supplied by P.P.H “STANLAB” Sp. z o.o. (Lublin, Poland). Glutaraldehyde was purchased from Warchem (Warsaw, Poland). All chemicals used were analytical grade and were used without further purification. 6-acetyloaminobutylo-9-chlorochino[3,2-b]benzo[ 1 , 4 ]thiazine (QBT) has been synthesized and purified in the Department of Organic Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, Poland, according to procedure published before [ 12 ]. 2.1 Absorption Measurements (Qualitative and Quantitative Studies) QBT was obtained according to the literature and its structure was confirmed using NMR and HR MS spectra [ 12 ]. To obtain a solution with a concentration of 2 · 10 − 5 mol · L − 1 , the QBT stock solution was prepared in DMSO and diluted in 0.05 mol · L − 1 phosphate buffer at pH 7.4. A quartz cuvette with a 10 mm pathlength was used to conduct the absorption spectrum of QBT using a JASCO V-760 spectrophotometer (Hachioji, Tokyo, Japan) with ± 1.5 nm wavelength repeatability. Absorption maxima were identified by recording absorption spectra within the 230–320 nm range. 2.2 Nanoparticles preparation and characterization In DMSO, a QBT solution with a concentration of 2 · 10 − 5 mol · L − 1 was prepared. HSA nanoparticles were synthesized through the desolvation method. After dissolving 20 mg of HSA in 2 mL of distilled water, dropwise additions of the previously made QBT solution were made. The magnetic stirrer was used to incubate and mix the samples at 550 rpm for 15 minutes. 8 mL of 96% ethanol was added to each sample following the incubation period. 11.8 µL of an 8% aqueous glutaraldehyde solution was used for crosslinking. The process was performed for 24 h using a magnetic stirrer at 550 rpm. The resulting suspension was centrifuged three times at 293 K and 15,000 rpm in distilled water to purify it, and it was then re-dispersed using a vortex and ultrasound. The nanoparticles were synthesized without the use of QBT, following the aforementioned procedure. All steps were performed at room temperature. Samples were lyophilized using Labconco FreeZone (A.G.A. Analytical, Warsaw, Poland). The concentration of QBT in the supernatant was determined by employing a standard calibration curve method to perform quantitative spectrophotometric determinations using the spectroscopic method. The amount of compound encapsulated in the nanoparticles was calculated using the following equation (Eq. ( 1 )): $$\:\text{E}\text{E}=\:\frac{\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{d}\text{r}\text{u}\text{g}\:\left(\text{m}\text{g}\right)-\text{f}\text{r}\text{e}\text{e}\:\text{d}\text{r}\text{u}\text{g}\:\left(\text{m}\text{g}\right)\:}{\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{d}\text{r}\text{u}\text{g}\:\left(\text{m}\text{g}\right)}\:\times\:100\text{\%}$$ 1 The morphology observation was conducted using a Nova Nano SEM 200 (FEI, Einthoven, Netherlands) ultra-high-resolution scanning electron microscope. Samples were placed at aluminum holders and coated with a 5 nm carbon layer (EM ACE600 sputter coater, Leica Microsystems, Wetzlar, Germany) then observed at an accelerated voltage of 10–15 kV range, under low vacuum (60 Pa), using Helix detector and an immersion mode. 2.3 In vitro drug release Separate beakers were used to mix nanoparticles with QBT with 60 mL of phosphate buffer (0.05 mol · L − 1 , pH 7.4). The mixture was kept at 310 K and stirred gently. Samples were collected at time intervals of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 24 and 25 h. Each sample was filtered through 0.22 µm syringe filters. A Jasco V-760 spectrophotometer (Hachioji, Tokyo, Japan) was used to measure the drug content of the medium using the UV-Vis spectrophotometric technique. To determine the in vitro kinetics and mechanisms of QBT release, the zero-order model (Eq. ( 2 )), first-order model (Eq. ( 3 )), the simplified Higuchi model (Eq. ( 4 )) and the Korsmeyer-Peppas model (Eq. ( 5 )) were used. $$\:{\text{C}}_{\text{r}}={\text{C}}_{0}+{\text{K}}_{0}\bullet\:\text{t}$$ 2 where Cr is the concentration of the released drug (mg · mL -1 ), C 0 is the initial concentration before the active release in time t (C 0 = 0), t is the release time in minutes and K 0 is the zero-order constant in units of 1 · time -1 [ 13 ]. First-order model was calculated using the following equation (Eq. ( 3 )): $$\:\frac{\text{d}\text{C}}{\text{d}\text{t}}=-{\text{K}}_{1}\bullet\:\text{C}$$ 3 where C is the concentration of drug (mg · mL -1 ) and K 1 is the first-order rate constant expressed in units of 1 · time -1 [ 13 ]. The simplified Higuchi model was calculated using the following equation (Eq. ( 4 )): $$\:\text{Q}={\text{K}}_{\text{H}}\sqrt{\text{t}}$$ 4 where Q is the concentration of the released drug (mg · mL -1 ), t is the release time in minutes and K H is the release constant of Higuchi [ 13 ]. The Korsmeyer-Peppas model was calculated using the following equation (Eq. ( 5 )): $$\:\frac{{\text{M}}_{\text{i}}}{{\text{M}}_{{\infty\:}}}={\text{K}}_{\text{K}\text{P}}{\text{t}}^{\text{n}}\:$$ 5 where \(\:\frac{{\text{M}}_{\text{i}}}{{\text{M}}_{{\infty\:}}}\) is the fractional solute release (mg · mL -1 ), t is the release time in minutes, K KP is the Korsmeyer-Peppas release constant and n is the diffusional exponent indicating the transport mechanism [ 13 , 14 ]. 2.4 Circular Dichroism Measurements Using a JASCO J-1500 spectropolarimeter (Hachioji, Japan), the far UV-CD spectra of HSA and HSA-QBT complexes at a [HSA]:[QBT] 1:4 molar ratio, as well as HSA-NPs and QBT-HSA-NPs, were recorded at a protein concentration of 0.5 · 10 − 6 mol · L − 1 . In an atmosphere of nitrogen at 293 K, the measurements were done in quartz cuvettes with a 1 mm optical path. The spectra were recorded in the wavelength range from 190 to 260 nm at wavelength intervals of 0.2 nm. The accuracy of the wavelength measurement was ± 0.1 nm, and the wavelength repeatability was ± 0.05 nm. Following the preparation process, the CD spectra of human serum albumin in the presence and absence of QBT as well as in nanoparticles were adjusted by subtracting the spectra obtained for phosphate buffer at pH 7.4, and smoothed using the Savitzky and Golay filters method [ 15 ]. Mean Residue Weight (Θ MRW ) for native HSA and HSA-NPs were calculated based on the following equation (Eq. ( 6 )): $$\:{{\Theta\:}}_{\text{M}\text{R}\text{W}}=\:\frac{\text{M}\text{R}\text{W}·{\Theta\:}\:}{10·\text{c}·\text{l}}$$ 6 where: Θ is the observed ellipticity for a given wavelength (deg), C is protein concentration (g · cm -3 ), l is the pathlength (cm), MRW is the mean residue weight (MRW HSA = 113.7 Da). ContinLL method and reference set 4 on the Dichroweb server has been used to calculate the α-helix percentage (%) content of HSA [ 16 ]. 2.5 Statistical analysis At least two repetitions were conducted for each sample. Utilizing the Spectra Manager Version 2.13.00 2002–2015 software, spectroscopic and spectrofluorescence spectra were examined. OriginPro version 8.5 SR1 software was used to vizualize the study's results as a mean ± relative standard deviation (SD). 3. Results 3.1 Absorption Measurements (Qualitative and Quantitative Studies The absorption spectrum of the sample with a concentration of 2 · 10 − 5 mol · L -1 was characterized by three distinct peaks (λ max 257 nm, λ max 285 nm, λ max 376 nm). The absorption spectrum has been shown in the Fig. 2 . The above absorption studies allowed for further quantitative investigations of the concentration of QBT encapsulated in the nanoparticles and the amount of drug released. 3.2 Nanoparticles preparation and characterization NPs were prepared according to the described procedure. The solutions of NPs that are obtained remain stable during centrifugation, vortexing, and ultrasonication. 97.44 ± 0.11% (n = 3; average ± SD) is the encapsulation efficiency of the QBT in the nanoparticles. Based on the data obtained with the scanning electron microscope (SEM), the surface of the nanoparticles can be described as smooth with no visible pores. The structures HSA-NPs named as A and QBT-HSA-NPs named as B have a similar spherical shape and do not differ significantly in size. The diameter of the nanoparticles was 92.680 ± 12.797 nm for HSA-NPs and 101.445 ± 9.907 nm for QBT-HSA-NPs. The visible difference can be noticed in reciprocal arrangement of spheres. Among the HSA-NPs spheres voids are present whereas, QBT-HSA-NPs are formed more densely. The particle size and morphology are shown in Fig. 3 . 3.3 In vitro drug release Using UV-Vis spectroscopy, the release of QBT from the nanoparticles was investigated. To replicate physiological conditions, phosphate buffer (pH 7.4) was employed and gently stirred at 310 K. The results are illustrated in Fig. 4 . The release of the substance started 30 min after the start of the test and was 1.28%. Following 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours, the drug concentration gradually increased to 2.56%, 3.68%, 4.83%, 5.92%, 6.96%, 8.21%, and 9.24%, respectively. The largest release of QBT, up to 11.34%, occurred 25 hours after the experiment began. Different models were fitted to the profiles recorded within the first 4 hours to ascertain the in vitro release kinetics of QBT. According to the regression coefficient (R 2 ) and rate constant values (K), the optimal kinetics model was determined. The results of the mathematical analysis have been shown in Table 1 . Table 1 Mathematical models of release kinetics of QBT from QBT-HSA-NPs. Zero-Order Model R 2 0.999 ± 0.070·10 − 5 K0 (1·h -1 ) 1.130 ± 0.313 First-Order Model R 2 0.966 ± 0.003 K1 (1·h -1 ) 0.181 ± 0.007 Higuchi Model R 2 0.984 ± 0.005 K H 3.091 ± 0.864 Korsmeyer-Peppas Model R 2 0.999 ± 7.070·10 − 5 K KP 0.186 ± 0.029 n 0.948 ± 0.022 In the absence and presence of QBT, circular dichroism (CD) was employed to ascertain changes in the secondary structure of HSA. Moreover, the secondary structure of HSA has been examined in relation to the preparation of nanoparticles. The results are shown in Figs. 5 and 6 . The data collected in Table 2 showed an increased packing of HSA at λ min 209 nm and λ min 220 nm, following the nanoparticle preparation process. This is evidenced by the decrease in Θ MRW , which was − 17 827.39 ± 355.91 deg · cm 2 · dmol − 1 and − 16 516.92 ± 286.79 deg · cm 2 · dmol − 1 for native HSA and − 3 943.03 ± 65.15 deg · cm 2 · dmol − 1 and − 3 746.05 ± 208.3 deg · cm 2 · dmol − 1 for HSA-NPs, respectively. Using ContinLL method and reference set 4 on the Dichroweb server [ 16 ] percentage (%) content of α-helix has been obtained. The α-helix content of native HSA and HSA in presence of QBT were 23.5 ± 0.009% and 9.25 ± 0.035%, respectively. In case of nanoparticles, % content of α-helix were 5.35 ± 0.017% for HSA-NPs and 19.38 ± 0.047% for QBT-HSA-NPs. Table 2 The mean residue ellipticity ([Θ]MRW) of HSA secondary structure elements. Θ MRW at 209 nm (deg · cm 2 · dmol − 1 ) Θ MRW at 220 nm (deg · cm 2 · dmol − 1 ) HSA -17 827.39 ± 355.91 -16 516.92 ± 286.79 HSA-NPs -3 943.03 ± 65.15 -3 746.05 ± 208.3 4. Discussion Due to the biocompatibility of HSA, it is considered an ideal biopolymer for the synthesis of modern drug delivery systems [ 17 ]. Phenothiazines show very high potential as antifungal and anticancer agents, as well as demonstrating efficacy in antiprotozoal therapies [ 18 ]. Since QBT can absorb in the UV-Vis range, spectroscopic analysis was done to find out the derivative's physicochemical characteristics. The UV-Vis spectroscopy method is the most often used method by scientists for both quantitative and qualitative analysis. This choice is driven by the straightforward procedure, excellent accuracy, or inexpensive analysis [ 19 , 20 ]. Figure 2 illustrates the UV-Vis spectrum of QBT, which is distinguished by three distinct maxima that potentially could have been employed in quantitative analyses. A single band is evident at λ max 257 nm, which is in accordance with the π-π* transition [ 21 ]. Two further bands are located at λ max 285 nm and λ max 376 nm. Our previous study [ 22 ], involving spectroscopic analysis of a thiazine derivative and its encapsulation attempt in bovine albumin nanoparticles, shows two distinct absorption bands. The first at λ max 265 nm and the second at λ max 315 nm. The differences may be due to differences in atomic structure. The absorption spectrum's distinctive appearance, which is a characteristic of each substance, is the cause of the variations in the distribution of absorption maxima and the appearance of the spectra [ 20 – 23 ]. The photochemical behavior of drugs was studied by Rodrigues et al. [ 24 ]. Spectroscopic studies showed an absorption spectrum of trifluoperazine that matches the shape of the spectrum in this work. The characteristic fingerprint of the compound is determined by the number of absorption maxima and the position of the individual bands. The similar spectral appearance is due to the presence of a structure – the thiazine ring [ 24 ]. The UV-Vis spectrophotometer is also an excellent device for assessing the amount of encapsulated drug. The main factors influencing the efficiency of this process are the selection of an appropriate preparation method and the functional groups present in the polymer structure [ 25 , 26 ]. The literature does not contain any data regarding the attempts to encapsulate QBT in albumin nanoparticles. The desolvation method achieved a QBT encapsulation efficiency of 97.44 ± 0.11%. In contrast, our previous research [ 22 ] on 10 H -2,7-diazaphenothiazine encapsulated in bovine albumin nanoparticles indicates an encapsulation efficiency of 66.67 ± 6.11%. Additionally, the work [ 25 ] involving 10-(2′-pyrimidyl)-3,6-diazaphenothiazine encapsulated in HSA nanoparticles demonstrated an EE value of 99.65 ± 0.05%. The desolvation method was also used in the aforementioned study, and the higher EE values may be due to structural differences in the phenothiazine derivatives analyzed. The preparation method and type of polymer or antisolvent used determine the size of the nanoparticles [ 26 ]. The dimension of the drug-containing nanoparticles was determined to be 101.445 ± 9.907 nm by SEM analysis, as illustrated in Fig. 3 . For example, the work of B von Storp et al. [ 27 ] focusing on HSA NPs made using the same method proved that the concentration and choice of solvent and dehydrating agent determines the size of the structures. Changing from ethanol to methanol allowed the researchers to significantly reduce the diameter, to sizes smaller than 70 nm [ 27 ]. Our previous studies have shown similar sizes of albumin NPs containing and without drugs. For example, NPs containing chlorambucil had a size in the order of 329.1 nm to 382.6 nm [ 28 ], and NPs with encapsulated 10 H -2,7-diazaphenothiazine had a size of 204 nm [ 22 ]. NPs without encapsulated drug had a size of 199.6 nm and 186 nm, respectively, as reference samples for nanoparticles with drugs. In the studies presented here, the diameter for HSA-NPs was 92.680 ± 12.797 nm. Once again, it can be seen that NPs with encapsulated drug substance have a larger size diameter than their reference group without drug [ 22 ]. From the study, it can be concluded that all albumin NPs are spherical which may ensure the uptake by target cells [ 29 ]. Aggregation of the NPs proceeds as a consequence of the compound's encapsulation, as demonstrated in our prior investigations [ 22 , 28 ]. The release of QBT from QBT-HSA-NPs proceeded in a two-step manner (Fig. 4 ). Mathematical models allow the release mechanism to be estimated as a function of the amount released versus time at the in vitro stage [ 13 ]. In order to better understand the release mechanism of QBT from QBT-HSA-NPs, the following models were used: zero-order, first-order, Higuchi and Korsmeyer-Peppas. Consistent with the values shown in Table 2 , QBT released according to the zero-order mechanism, via QBT diffusion and HSA swelling (0.43 < n < 0.85) [ 13 , 30 ]. It was possible to estimate the same mechanism in almost all of our previous studies using the Korsmeyer-Peppas model [ 28 , 31 , 32 ]. Wang et al. [ 33 ] conducted a study in which the release of paclitaxel from BSA NPs incorporated into an o-phthalaldehyde-terminated 4-armed poly(ethylene glycol) hydrogel was modeled using the Korsmeyer-Peppas model. The results indicated that paclitaxel release was influenced by polymer relaxation and drug diffusion. One of the greatest methods for analyzing how chemicals affect proteins' secondary structures and understanding ligand-protein interactions is circular dichroism (CD) spectroscopy. Additionally, it facilitates the quantification of ligand binding [ 15 ]. The results of applying the previously mentioned method to examine the impact of QBT on HSA are displayed in Figs. 5 and 6 . It was concluded from the study that the interaction between QBT and HSA is accompanied by a modification in the protein's secondary structure. Basically, the molecule changes the amount of α-helix in HSA. In comparison to native HSA, the percentage of α-helix decreased in HSA in the presence of QBT. Nevertheless, the quantity of α-helices that are preserved in QBT-HSA-NPs nanoparticles is less than that of HSA-NPs due to the presence of QBT. The drug's side effects may be impacted by the destabilization of HSA's secondary structure in the presence of QBT, which also serves as justification for trying to encapsulate QBT in nanoparticle. The CD spectra of HSA-NPs and QBT-HSA-NPs are shown in Figs. 5 and 6 . As can be seen, the presence of QBT modifies the spectral pattern when bound to HSA, but again a correlation can be seen that HSA lost its secondary structure after NPs preparation, whereas encapsulation of QBT results in partial preservation of the secondary structure. A study by Owczarzy et al. [ 34 ] on 9-fluoro-5-alkyl-12(H)-chino[3,4-b][ 1 , 4 ]benzothiazine chloride demonstrated a lack of effect on the secondary structure of HSA, which may be due to its ability to absorb other wavelengths or the presence of other substituents in the molecule. As described in our previous works [ 22 , 28 , 31 , 32 ], drug encapsulation can have a protective effect on changes in albumin chain packing (Table 2 ) and modifications of the secondary structure. Apart from our study, few authors have used CD in the analysis of polymer structure changes after preparation of nanoparticles or other materials [ 35 – 38 ]. The observed changes in the structure of HSA influenced by the presence of QBT at the molecular level, may not have a strong influence on the side effects generated in the in vivo system, but further studies are required to unequivocally support this statement. Conclusions The research is a continuation of the attempt to encapsulation new quinobenzothiazine derivative into nanoparticles. Also, it allowed to analyze a quinobenzothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[ 1 , 4 ]thiazine) with potential anticancer activity using spectroscopic techniques and to attempt to encapsulate the compound in human albumin nanoparticles. Following the synthesis, particles with a diameter of 92.680 nm were obtained, and their diameter increased to 110.445 nm following the encapsulation process. Spectroscopic studies confirmed the presence of encapsulated QBT, which also allowed release studies to be performed. In order to ascertain the release kinetics mechanism, which is essential for the particles' medical applications, mathematical methods were implemented. In a biological environment, the nanocarriers' activity can be assessed through the release kinetics calculations and the graph of QBT release from the nanoparticles. The above results indicate the potential applicability of QBT-HSA-NPs in medicine. Due to the insufficiency of studies on QBT-containing nanoparticles, regardless of the promising results, further studies on albumin nanoparticles with QBT are required to further investigate the therapeutic potency of the structures formed. They can also form the basis for further preclinical and clinical studies. Declarations Funding Sources This research was funded by the Medical University of Silesia, grant numbers: BNW-2-048/K/4/F, BNW-2-028/K/5/F, BNW-1-009/K/4/F, BNW-2-049/K/4/F, BNW-2-110/K/4/F. ACKNOWLEDGMENT The authors declare no conflict of interest. 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Pharmaceuticals (Basel) 16:14–23. https://doi.org/10.3390/ph16101423 Wang T, Ding J, Chen Z, Zhang Z, Rong Y, Li G, He C, Chen X (2024) Injectable, adhesive albumin nanoparticle-incorporated hydrogel for sustained localized drug delivery and efficient tumor treatment. ACS Appl Mater Interfaces 16(8):9868–9879. https://doi.org/10.1021/acsami.3c18306 Owczarzy A, Rogóż W, Kulig K, Pożycka J, Zięba A, Maciążek-Jurczyk M (2023) Spectroscopic studies of quinobenzothiazine derivative in terms of the in vitro interaction with selected human plasma proteins: Part 2. Molecules 28(2):698. https://doi.org/10.3390/molecules28020698 Ciepluch K, Biehl R, Bryszewska M, Arabski M (2020) Poly(propylene imine) dendrimers can bind to PEGylated albumin at PEG and albumin surface: Biophysical examination of a PEGylated platform to transport cationic dendritic nanoparticles. Biopolymers 111(9):e23386. https://doi.org/10.1002/bip.23386 Morozova OV, Pavlova ER, Bagrov DV, Barinov NA, Prusakov KA, Isaeva EI, Podgorsky VV, Basmanov DV, Klinov DV (2018) Protein nanoparticles with ligand-binding and enzymatic activities. Int J Nanomedicine 13:6637–6646. https://doi.org/10.2147/IJN.S177627 Varytis P, Stefanou N, Christofi A, Papanikolaou N (2015) Strong circular dichroism of core-shell magnetoplasmonic nanoparticles. J Opt Soc Am B 32:1063–1069. https://doi.org/10.1364/JOSAB.32.001063 Turner GA, Dunlap CE, Higgins AJ, Simpson GJ (2025) Dark-field absorbance circular dichroism of oriented chiral thin films. J Phys Chem Lett 30:1403–1408. https://doi.org/10.1021/acs.jpclett.4c02984 Additional Declarations No competing interests reported. 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Katowice","correspondingAuthor":false,"prefix":"","firstName":"Patrycja","middleName":"","lastName":"Sarkowicz","suffix":""},{"id":476138958,"identity":"5b48b319-f49f-498c-a8e7-a7953a10803a","order_by":2,"name":"Małgorzata Jeleń","email":"","orcid":"","institution":"Medical University of Silesia in Katowice","correspondingAuthor":false,"prefix":"","firstName":"Małgorzata","middleName":"","lastName":"Jeleń","suffix":""},{"id":476138959,"identity":"7477989d-b778-4dc1-aa1e-9e50ee72bbe1","order_by":3,"name":"Beata Morak-Młodawska","email":"","orcid":"","institution":"Medical University of Silesia in Katowice","correspondingAuthor":false,"prefix":"","firstName":"Beata","middleName":"","lastName":"Morak-Młodawska","suffix":""},{"id":476138960,"identity":"9e6904d3-d676-4d72-afce-8f4cd9965665","order_by":4,"name":"Magdalena Ziąbka","email":"","orcid":"","institution":"AGH University of Krakow","correspondingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Ziąbka","suffix":""},{"id":476138963,"identity":"8ea3a78c-b3fc-440f-b3a4-ee8c9dd83e31","order_by":5,"name":"Aleksandra Owczarzy","email":"","orcid":"","institution":"Medical University of Silesia in Katowice","correspondingAuthor":false,"prefix":"","firstName":"Aleksandra","middleName":"","lastName":"Owczarzy","suffix":""},{"id":476138964,"identity":"98746ee1-31bb-490c-94d5-a34ed42c95ad","order_by":6,"name":"Wojciech Rogóż","email":"","orcid":"","institution":"Medical University of Silesia in Katowice","correspondingAuthor":false,"prefix":"","firstName":"Wojciech","middleName":"","lastName":"Rogóż","suffix":""},{"id":476138965,"identity":"f27d7e63-d9b2-4d75-b2a6-c98ef3d0c1cd","order_by":7,"name":"Małgorzata 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10:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6749623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6749623/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85480210,"identity":"d8601e3b-be94-4c59-b0bf-57e92614023f","added_by":"auto","created_at":"2025-06-26 10:51:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5926,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structure of QBT.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/8bd998ceb717bb2674578fdd.png"},{"id":85481016,"identity":"c01a6822-189b-460e-ad5f-2b4aa53e870f","added_by":"auto","created_at":"2025-06-26 10:59:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10333,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption spectrum of QBT solution.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/5c0a83e5967b537f6e14aeb2.png"},{"id":85481017,"identity":"7b8c3f5d-bb47-4dd0-be47-34afc74d4a46","added_by":"auto","created_at":"2025-06-26 10:59:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":499834,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM microscope data of the (A) HSA-NPs and (B) for QBT-HSA-NPs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/d108a3b9583eeff31dda9482.png"},{"id":85480211,"identity":"55ab4e00-637d-4123-bc8f-0819f4060b4b","added_by":"auto","created_at":"2025-06-26 10:51:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14097,"visible":true,"origin":"","legend":"\u003cp\u003eQBT release from QBT-HSA-NPs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/f84523bc3d273729105b86ab.png"},{"id":85480214,"identity":"eefe9430-6da4-4853-8231-8b66046d1251","added_by":"auto","created_at":"2025-06-26 10:51:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12093,"visible":true,"origin":"","legend":"\u003cp\u003eCD spectra of HSA-QBT and HSA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/c04eb7ef3cc3d44e5cb5c34c.png"},{"id":85480217,"identity":"f29da0ab-b4e6-4209-a8c9-54f6138328f3","added_by":"auto","created_at":"2025-06-26 10:51:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14025,"visible":true,"origin":"","legend":"\u003cp\u003eCD spectra of QBT-HSA-NPs and HSA-NPs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/536893713d87e76ba686e6bb.png"},{"id":85482441,"identity":"e94d50e9-5aac-47df-99af-8d899aab3ece","added_by":"auto","created_at":"2025-06-26 11:15:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1263204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6749623/v1/1120df09-bc3d-4f3e-b26b-95dcb3d076d1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spectroscopic analysis of human serum albumin nanoparticles with encapsulated phenothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine) – continuation studies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanomaterials are one of the most rapidly evolving research topics. It is an interdisciplinary science field, the understanding of which requires knowledge from fields such as biology, chemistry, electronics physics or engineering [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nanomaterials are one of the most rapidly evolving research topics. It is an interdisciplinary science field, the understanding of which requires knowledge from fields such as biology, chemistry, electronics physics or engineering [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Numerous studies show nanoparticles (NPs) as effective tools in many areas of biological and other sciences. They are used as drug carriers in both therapeutics and diagnostics. Their physicochemical properties, ability to bind the drug and release the therapeutic substance and low toxicity determine the clinical application of NPs. They are characterised by sizes in the range of 1-100 nm and their properties depend on the size and surface functionalization. The growing interest in nanostructures is consolidated by their ability to be imaged utilizing microscopic methods, such as scanning tunneling microscopy (STM), scanning transmission electron microscopy (STEM), or tandem electron microscopy (TEM) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NPs' fate in the body is significantly influenced by their physicochemical properties, which significantly alter absorption, pharmacokinetics, clearance, cellular uptake, and cytotoxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe chemical structure of NPs allows for the classification of them into three groups: organic, inorganic, and carbon nanoparticles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The polymer that is the building block of the NPs as a drug delivery system, must follow several requirements. Firstly, it must be biodegradable or completely eliminated from the body in a timeframe that allows repeated administration without the danger of accumulation in the body. It is imperative that neither the polymer nor the product of its degradation be immunogenic or toxic. In addition, it should allow the synthesis of nanoparticles with appropriate properties in relation to the drug delivery target for which the nanoparticles have been designed [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Polymer NPs can serve as carriers for biologically active particles adsorbed on the surface or encapsulated inside them. Both natural and synthetic polymers can be used for synthesis. Natural polymers are recommended for the synthesis of nanoparticles due to their biodegradability. Advantages of polymeric NPs include high drug encapsulation efficiency, higher intracellular uptake compared to other drug carriers, higher stability of active substances, biocompatibility, and biodegradability. On the other hand, disadvantages include high production costs, risk of non-biodegradability, toxicity of solvents used for synthesis or brittleness [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuman serum albumin (HSA) was first used in the delivery of paclitaxel in the medication Abraxane. Early in 2005, it was introduced to the medical community [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Its easy availability, biocompatibility and ability to accumulate in tissues with a high metabolism have determined that albumin has become the most widely used natural polymer for the synthesis of therapeutic nanoparticles. The Federal Food and Drug Administration (FDA) has approved HSA for this purpose. It is a particularly attractive carrier for anti-cancer and anti-inflammatory drugs. Tumor-affected tissue is characterized by increased expression of the glycoprotein \u0026ndash; secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin, which results in the accumulation of albumin and thus increased efficacy of cytostatic drugs encapsulated in protein nanoparticles [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, HSA is one of the natural antioxidants which may have a role in increasing the efficacy of the nanoparticle [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhenothiazines are a group of compounds with a broad spectrum of biological activities, including the following antipsychotic, antihistamine, antitussive and antiemetic. Also documented for this group of compounds are anticancer, antimicrobial and, related, against multidrug resistance (MDR) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e6-Acetylaminobutyl-9-chloroquino[3,2-b]benzo[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]thiazine (QBT) is a four-ring phenothiazine derivative obtained by replacing one benzene ring with a quinoline system and introducing an acetylaminobutyl substituent to the thiazine nitrogen atom. QBT potently inhibited phytohemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells (PBMC), tumor necrosis alpha production, and growth of tumor cell lines. QBT shows comparable activity to cis-platinum against cancer cell lines A-431, L-1210 and SW-948. QBT proved to be suppressive in the models of delayed type hypersensitivity to ovalbumin and carrageenan induced footpad tests in mice. This compound was also effective in the amelioration of contact sensitivity to oxazolone and experimentally induced psoriasis. In \u003cem\u003ein vivo\u003c/em\u003e mouse studies, the efficacy of the compound QBT in alleviating dextran sulphate sodium (DSS)-induced ulcerative colitis has been demonstrated [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The potential drug substance used in this research \u0026ndash; QBT, is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe aim of this novelty study was to perform spectroscopic analysis of QBT and its encapsulation into HSA nanoparticles. The research conducted presents the use of QBT as an encapsulated substance in albumin nanoparticles prepared using the desolvation method, release study and confirmation of the above results by spectroscopic techniques. Despite reports on albumin nanoparticles and QBT, no one has published studies on QBT encapsulation in nanoparticles to data. The work also continues our series of studies on the encapsulation of phenothiazine derivatives in albumin nanoparticles.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eDmethyl sulfoxide (DMSO) and human serum albumin (HSA) with a minimum purity of 96% were acquired from Sigma Aldrich in Steinheim, Germany. Ethanol was supplied by P.P.H \u0026ldquo;STANLAB\u0026rdquo; Sp. z o.o. (Lublin, Poland). Glutaraldehyde was purchased from Warchem (Warsaw, Poland). All chemicals used were analytical grade and were used without further purification. 6-acetyloaminobutylo-9-chlorochino[3,2-b]benzo[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]thiazine (QBT) has been synthesized and purified in the Department of Organic Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, Poland, according to procedure published before [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Absorption Measurements (Qualitative and Quantitative Studies)\u003c/h2\u003e \u003cp\u003eQBT was obtained according to the literature and its structure was confirmed using NMR and HR MS spectra [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To obtain a solution with a concentration of 2 \u0026middot; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the QBT stock solution was prepared in DMSO and diluted in 0.05 mol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e phosphate buffer at pH 7.4. A quartz cuvette with a 10 mm pathlength was used to conduct the absorption spectrum of QBT using a JASCO V-760 spectrophotometer (Hachioji, Tokyo, Japan) with \u0026plusmn;\u0026thinsp;1.5 nm wavelength repeatability. Absorption maxima were identified by recording absorption spectra within the 230\u0026ndash;320 nm range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Nanoparticles preparation and characterization\u003c/h2\u003e \u003cp\u003eIn DMSO, a QBT solution with a concentration of 2 \u0026middot; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was prepared. HSA nanoparticles were synthesized through the desolvation method. After dissolving 20 mg of HSA in 2 mL of distilled water, dropwise additions of the previously made QBT solution were made. The magnetic stirrer was used to incubate and mix the samples at 550 rpm for 15 minutes. 8 mL of 96% ethanol was added to each sample following the incubation period. 11.8 \u0026micro;L of an 8% aqueous glutaraldehyde solution was used for crosslinking. The process was performed for 24 h using a magnetic stirrer at 550 rpm. The resulting suspension was centrifuged three times at 293 K and 15,000 rpm in distilled water to purify it, and it was then re-dispersed using a vortex and ultrasound. The nanoparticles were synthesized without the use of QBT, following the aforementioned procedure. All steps were performed at room temperature. Samples were lyophilized using Labconco FreeZone (A.G.A. Analytical, Warsaw, Poland).\u003c/p\u003e \u003cp\u003eThe concentration of QBT in the supernatant was determined by employing a standard calibration curve method to perform quantitative spectrophotometric determinations using the spectroscopic method. The amount of compound encapsulated in the nanoparticles was calculated using the following equation (Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{E}=\\:\\frac{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{d}\\text{r}\\text{u}\\text{g}\\:\\left(\\text{m}\\text{g}\\right)-\\text{f}\\text{r}\\text{e}\\text{e}\\:\\text{d}\\text{r}\\text{u}\\text{g}\\:\\left(\\text{m}\\text{g}\\right)\\:}{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{d}\\text{r}\\text{u}\\text{g}\\:\\left(\\text{m}\\text{g}\\right)}\\:\\times\\:100\\text{\\%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe morphology observation was conducted using a Nova Nano SEM 200 (FEI, Einthoven, Netherlands) ultra-high-resolution scanning electron microscope. Samples were placed at aluminum holders and coated with a 5 nm carbon layer (EM ACE600 sputter coater, Leica Microsystems, Wetzlar, Germany) then observed at an accelerated voltage of 10\u0026ndash;15 kV range, under low vacuum (60 Pa), using Helix detector and an immersion mode.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 \u003cem\u003eIn vitro\u003c/em\u003e drug release\u003c/h2\u003e \u003cp\u003eSeparate beakers were used to mix nanoparticles with QBT with 60 mL of phosphate buffer (0.05 mol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, pH 7.4). The mixture was kept at 310 K and stirred gently. Samples were collected at time intervals of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 24 and 25 h. Each sample was filtered through 0.22 \u0026micro;m syringe filters. A Jasco V-760 spectrophotometer (Hachioji, Tokyo, Japan) was used to measure the drug content of the medium using the UV-Vis spectrophotometric technique.\u003c/p\u003e \u003cp\u003eTo determine the \u003cem\u003ein vitro\u003c/em\u003e kinetics and mechanisms of QBT release, the zero-order model (Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)), first-order model (Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)), the simplified Higuchi model (Eq.\u0026nbsp;(\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)) and the Korsmeyer-Peppas model (Eq.\u0026nbsp;(\u003cspan refid=\"Equ5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)) were used.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:{\\text{C}}_{\\text{r}}={\\text{C}}_{0}+{\\text{K}}_{0}\\bullet\\:\\text{t}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere Cr is the concentration of the released drug (mg \u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e), C\u003csub\u003e0\u003c/sub\u003e is the initial concentration before the active release in time t (C\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0), t is the release time in minutes and K\u003csub\u003e0\u003c/sub\u003e is the zero-order constant in units of 1 \u0026middot; time\u003csup\u003e-1\u003c/sup\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFirst-order model was calculated using the following equation (Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)):\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\frac{\\text{d}\\text{C}}{\\text{d}\\text{t}}=-{\\text{K}}_{1}\\bullet\\:\\text{C}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere C is the concentration of drug (mg \u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e) and K\u003csub\u003e1\u003c/sub\u003e is the first-order rate constant expressed in units of 1 \u0026middot; time\u003csup\u003e-1\u003c/sup\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe simplified Higuchi model was calculated using the following equation (Eq.\u0026nbsp;(\u003cspan refid=\"Equ4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)):\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:\\text{Q}={\\text{K}}_{\\text{H}}\\sqrt{\\text{t}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere Q is the concentration of the released drug (mg \u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e), t is the release time in minutes and K\u003csub\u003eH\u003c/sub\u003e is the release constant of Higuchi [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Korsmeyer-Peppas model was calculated using the following equation (Eq.\u0026nbsp;(\u003cspan refid=\"Equ5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)):\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:\\frac{{\\text{M}}_{\\text{i}}}{{\\text{M}}_{{\\infty\\:}}}={\\text{K}}_{\\text{K}\\text{P}}{\\text{t}}^{\\text{n}}\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{\\text{M}}_{\\text{i}}}{{\\text{M}}_{{\\infty\\:}}}\\)\u003c/span\u003e\u003c/span\u003e is the fractional solute release (mg \u0026middot; mL\u003csup\u003e-1\u003c/sup\u003e), t is the release time in minutes, K\u003csub\u003eKP\u003c/sub\u003e is the Korsmeyer-Peppas release constant and n is the diffusional exponent indicating the transport mechanism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Circular Dichroism Measurements\u003c/h2\u003e \u003cp\u003eUsing a JASCO J-1500 spectropolarimeter (Hachioji, Japan), the far UV-CD spectra of HSA and HSA-QBT complexes at a [HSA]:[QBT] 1:4 molar ratio, as well as HSA-NPs and QBT-HSA-NPs, were recorded at a protein concentration of 0.5 \u0026middot; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e mol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In an atmosphere of nitrogen at 293 K, the measurements were done in quartz cuvettes with a 1 mm optical path. The spectra were recorded in the wavelength range from 190 to 260 nm at wavelength intervals of 0.2 nm. The accuracy of the wavelength measurement was \u0026plusmn;\u0026thinsp;0.1 nm, and the wavelength repeatability was \u0026plusmn;\u0026thinsp;0.05 nm. Following the preparation process, the CD spectra of human serum albumin in the presence and absence of QBT as well as in nanoparticles were adjusted by subtracting the spectra obtained for phosphate buffer at pH 7.4, and smoothed using the Savitzky and Golay filters method [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMean Residue Weight (Θ\u003csub\u003eMRW\u003c/sub\u003e) for native HSA and HSA-NPs were calculated based on the following equation (Eq.\u0026nbsp;(\u003cspan refid=\"Equ6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)):\u003cdiv id=\"Equ6\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ6\" name=\"EquationSource\"\u003e\n$$\\:{{\\Theta\\:}}_{\\text{M}\\text{R}\\text{W}}=\\:\\frac{\\text{M}\\text{R}\\text{W}\u0026middot;{\\Theta\\:}\\:}{10\u0026middot;\\text{c}\u0026middot;\\text{l}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere: Θ is the observed ellipticity for a given wavelength (deg), C is protein concentration (g \u0026middot; cm\u003csup\u003e-3\u003c/sup\u003e), l is the pathlength (cm), MRW is the mean residue weight (MRW\u003csub\u003eHSA\u003c/sub\u003e = 113.7 Da).\u003c/p\u003e \u003cp\u003eContinLL method and reference set 4 on the Dichroweb server has been used to calculate the α-helix percentage (%) content of HSA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eAt least two repetitions were conducted for each sample. Utilizing the Spectra Manager Version 2.13.00 2002\u0026ndash;2015 software, spectroscopic and spectrofluorescence spectra were examined. OriginPro version 8.5 SR1 software was used to vizualize the study's results as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;relative standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Absorption Measurements (Qualitative and Quantitative Studies\u003c/h2\u003e \u003cp\u003eThe absorption spectrum of the sample with a concentration of 2 \u0026middot; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol \u0026middot; L\u003csup\u003e-1\u003c/sup\u003e was characterized by three distinct peaks (λ\u003csub\u003emax\u003c/sub\u003e 257 nm, λ\u003csub\u003emax\u003c/sub\u003e 285 nm, λ\u003csub\u003emax\u003c/sub\u003e 376 nm). The absorption spectrum has been shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe above absorption studies allowed for further quantitative investigations of the concentration of QBT encapsulated in the nanoparticles and the amount of drug released.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Nanoparticles preparation and characterization\u003c/h2\u003e \u003cp\u003eNPs were prepared according to the described procedure. The solutions of NPs that are obtained remain stable during centrifugation, vortexing, and ultrasonication. 97.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11% (n\u0026thinsp;=\u0026thinsp;3; average\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) is the encapsulation efficiency of the QBT in the nanoparticles.\u003c/p\u003e \u003cp\u003eBased on the data obtained with the scanning electron microscope (SEM), the surface of the nanoparticles can be described as smooth with no visible pores. The structures HSA-NPs named as A and QBT-HSA-NPs named as B have a similar spherical shape and do not differ significantly in size. The diameter of the nanoparticles was 92.680\u0026thinsp;\u0026plusmn;\u0026thinsp;12.797 nm for HSA-NPs and 101.445\u0026thinsp;\u0026plusmn;\u0026thinsp;9.907 nm for QBT-HSA-NPs. The visible difference can be noticed in reciprocal arrangement of spheres. Among the HSA-NPs spheres voids are present whereas, QBT-HSA-NPs are formed more densely. The particle size and morphology are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 \u003cem\u003eIn vitro\u003c/em\u003e drug release\u003c/h2\u003e \u003cp\u003eUsing UV-Vis spectroscopy, the release of QBT from the nanoparticles was investigated. To replicate physiological conditions, phosphate buffer (pH 7.4) was employed and gently stirred at 310 K. The results are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The release of the substance started 30 min after the start of the test and was 1.28%. Following 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours, the drug concentration gradually increased to 2.56%, 3.68%, 4.83%, 5.92%, 6.96%, 8.21%, and 9.24%, respectively. The largest release of QBT, up to 11.34%, occurred 25 hours after the experiment began.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDifferent models were fitted to the profiles recorded within the first 4 hours to ascertain the \u003cem\u003ein vitro\u003c/em\u003e release kinetics of QBT. According to the regression coefficient (R\u003csup\u003e2\u003c/sup\u003e) and rate constant values (K), the optimal kinetics model was determined. The results of the mathematical analysis have been shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMathematical models of release kinetics of QBT from QBT-HSA-NPs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eZero-Order\u003c/p\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.999\u0026thinsp;\u0026plusmn;\u0026thinsp;0.070\u0026middot;10\u0026thinsp;\u0026minus;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK0 (1\u0026middot;h\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.130\u0026thinsp;\u0026plusmn;\u0026thinsp;0.313\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFirst-Order\u003c/p\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.966\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK1 (1\u0026middot;h\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.181\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHiguchi\u003c/p\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.984\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.091\u0026thinsp;\u0026plusmn;\u0026thinsp;0.864\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eKorsmeyer-Peppas\u003c/p\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.999\u0026thinsp;\u0026plusmn;\u0026thinsp;7.070\u0026middot;10\u0026thinsp;\u0026minus;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK\u003csub\u003eKP\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.186\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.948\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the absence and presence of QBT, circular dichroism (CD) was employed to ascertain changes in the secondary structure of HSA. Moreover, the secondary structure of HSA has been examined in relation to the preparation of nanoparticles. The results are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe data collected in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed an increased packing of HSA at λ\u003csub\u003emin\u003c/sub\u003e 209 nm and λ\u003csub\u003emin\u003c/sub\u003e 220 nm, following the nanoparticle preparation process. This is evidenced by the decrease in Θ\u003csub\u003eMRW\u003c/sub\u003e, which was \u0026minus;\u0026thinsp;17 827.39\u0026thinsp;\u0026plusmn;\u0026thinsp;355.91 deg \u0026middot; cm\u003csup\u003e2\u003c/sup\u003e \u0026middot; dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u0026minus;\u0026thinsp;16 516.92\u0026thinsp;\u0026plusmn;\u0026thinsp;286.79 deg \u0026middot; cm\u003csup\u003e2\u003c/sup\u003e \u0026middot; dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for native HSA and \u0026minus;\u0026thinsp;3 943.03\u0026thinsp;\u0026plusmn;\u0026thinsp;65.15 deg \u0026middot; cm\u003csup\u003e2\u003c/sup\u003e \u0026middot; dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u0026minus;\u0026thinsp;3 746.05\u0026thinsp;\u0026plusmn;\u0026thinsp;208.3 deg \u0026middot; cm\u003csup\u003e2\u003c/sup\u003e \u0026middot; dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for HSA-NPs, respectively.\u003c/p\u003e \u003cp\u003eUsing ContinLL method and reference set 4 on the Dichroweb server [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] percentage (%) content of α-helix has been obtained. The α-helix content of native HSA and HSA in presence of QBT were 23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009% and 9.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035%, respectively. In case of nanoparticles, % content of α-helix were 5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017% for HSA-NPs and 19.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047% for QBT-HSA-NPs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe mean residue ellipticity ([Θ]MRW) of HSA secondary structure elements.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΘ\u003csub\u003eMRW\u003c/sub\u003e at 209 nm\u003c/p\u003e \u003cp\u003e(deg \u0026middot; cm\u003csup\u003e2\u003c/sup\u003e \u0026middot; dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΘ\u003csub\u003eMRW\u003c/sub\u003e at 220 nm\u003c/p\u003e \u003cp\u003e(deg \u0026middot; cm\u003csup\u003e2\u003c/sup\u003e \u0026middot; dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-17\u0026nbsp;827.39\u0026thinsp;\u0026plusmn;\u0026thinsp;355.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-16\u0026nbsp;516.92\u0026thinsp;\u0026plusmn;\u0026thinsp;286.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSA-NPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-3\u0026nbsp;943.03\u0026thinsp;\u0026plusmn;\u0026thinsp;65.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-3\u0026nbsp;746.05\u0026thinsp;\u0026plusmn;\u0026thinsp;208.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDue to the biocompatibility of HSA, it is considered an ideal biopolymer for the synthesis of modern drug delivery systems [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Phenothiazines show very high potential as antifungal and anticancer agents, as well as demonstrating efficacy in antiprotozoal therapies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince QBT can absorb in the UV-Vis range, spectroscopic analysis was done to find out the derivative's physicochemical characteristics. The UV-Vis spectroscopy method is the most often used method by scientists for both quantitative and qualitative analysis. This choice is driven by the straightforward procedure, excellent accuracy, or inexpensive analysis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the UV-Vis spectrum of QBT, which is distinguished by three distinct maxima that potentially could have been employed in quantitative analyses. A single band is evident at λ\u003csub\u003emax\u003c/sub\u003e 257 nm, which is in accordance with the π-π* transition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Two further bands are located at λ\u003csub\u003emax\u003c/sub\u003e 285 nm and λ\u003csub\u003emax\u003c/sub\u003e 376 nm. Our previous study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], involving spectroscopic analysis of a thiazine derivative and its encapsulation attempt in bovine albumin nanoparticles, shows two distinct absorption bands. The first at λ\u003csub\u003emax\u003c/sub\u003e 265 nm and the second at λ\u003csub\u003emax\u003c/sub\u003e 315 nm. The differences may be due to differences in atomic structure. The absorption spectrum's distinctive appearance, which is a characteristic of each substance, is the cause of the variations in the distribution of absorption maxima and the appearance of the spectra [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The photochemical behavior of drugs was studied by Rodrigues et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Spectroscopic studies showed an absorption spectrum of trifluoperazine that matches the shape of the spectrum in this work. The characteristic fingerprint of the compound is determined by the number of absorption maxima and the position of the individual bands. The similar spectral appearance is due to the presence of a structure – the thiazine ring [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe UV-Vis spectrophotometer is also an excellent device for assessing the amount of encapsulated drug. The main factors influencing the efficiency of this process are the selection of an appropriate preparation method and the functional groups present in the polymer structure [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The literature does not contain any data regarding the attempts to encapsulate QBT in albumin nanoparticles. The desolvation method achieved a QBT encapsulation efficiency of 97.44 ± 0.11%. In contrast, our previous research [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] on 10\u003cem\u003eH\u003c/em\u003e-2,7-diazaphenothiazine encapsulated in bovine albumin nanoparticles indicates an encapsulation efficiency of 66.67 ± 6.11%. Additionally, the work [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] involving 10-(2′-pyrimidyl)-3,6-diazaphenothiazine encapsulated in HSA nanoparticles demonstrated an EE value of 99.65 ± 0.05%. The desolvation method was also used in the aforementioned study, and the higher EE values may be due to structural differences in the phenothiazine derivatives analyzed.\u003c/p\u003e \u003cp\u003eThe preparation method and type of polymer or antisolvent used determine the size of the nanoparticles [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The dimension of the drug-containing nanoparticles was determined to be 101.445 ± 9.907 nm by SEM analysis, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. For example, the work of B von Storp et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] focusing on HSA NPs made using the same method proved that the concentration and choice of solvent and dehydrating agent determines the size of the structures. Changing from ethanol to methanol allowed the researchers to significantly reduce the diameter, to sizes smaller than 70 nm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our previous studies have shown similar sizes of albumin NPs containing and without drugs. For example, NPs containing chlorambucil had a size in the order of 329.1 nm to 382.6 nm [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and NPs with encapsulated 10\u003cem\u003eH\u003c/em\u003e-2,7-diazaphenothiazine had a size of 204 nm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. NPs without encapsulated drug had a size of 199.6 nm and 186 nm, respectively, as reference samples for nanoparticles with drugs. In the studies presented here, the diameter for HSA-NPs was 92.680 ± 12.797 nm. Once again, it can be seen that NPs with encapsulated drug substance have a larger size diameter than their reference group without drug [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. From the study, it can be concluded that all albumin NPs are spherical which may ensure the uptake by target cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Aggregation of the NPs proceeds as a consequence of the compound's encapsulation, as demonstrated in our prior investigations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe release of QBT from QBT-HSA-NPs proceeded in a two-step manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Mathematical models allow the release mechanism to be estimated as a function of the amount released versus time at the \u003cem\u003ein vitro\u003c/em\u003e stage [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In order to better understand the release mechanism of QBT from QBT-HSA-NPs, the following models were used: zero-order, first-order, Higuchi and Korsmeyer-Peppas. Consistent with the values shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, QBT released according to the zero-order mechanism, via QBT diffusion and HSA swelling (0.43 \u0026lt; n \u0026lt; 0.85) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It was possible to estimate the same mechanism in almost all of our previous studies using the Korsmeyer-Peppas model [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Wang et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] conducted a study in which the release of paclitaxel from BSA NPs incorporated into an o-phthalaldehyde-terminated 4-armed poly(ethylene glycol) hydrogel was modeled using the Korsmeyer-Peppas model. The results indicated that paclitaxel release was influenced by polymer relaxation and drug diffusion.\u003c/p\u003e \u003cp\u003eOne of the greatest methods for analyzing how chemicals affect proteins' secondary structures and understanding ligand-protein interactions is circular dichroism (CD) spectroscopy. Additionally, it facilitates the quantification of ligand binding [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The results of applying the previously mentioned method to examine the impact of QBT on HSA are displayed in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. It was concluded from the study that the interaction between QBT and HSA is accompanied by a modification in the protein's secondary structure. Basically, the molecule changes the amount of α-helix in HSA. In comparison to native HSA, the percentage of α-helix decreased in HSA in the presence of QBT. Nevertheless, the quantity of α-helices that are preserved in QBT-HSA-NPs nanoparticles is less than that of HSA-NPs due to the presence of QBT. The drug's side effects may be impacted by the destabilization of HSA's secondary structure in the presence of QBT, which also serves as justification for trying to encapsulate QBT in nanoparticle. The CD spectra of HSA-NPs and QBT-HSA-NPs are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As can be seen, the presence of QBT modifies the spectral pattern when bound to HSA, but again a correlation can be seen that HSA lost its secondary structure after NPs preparation, whereas encapsulation of QBT results in partial preservation of the secondary structure. A study by Owczarzy et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] on 9-fluoro-5-alkyl-12(H)-chino[3,4-b][\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]benzothiazine chloride demonstrated a lack of effect on the secondary structure of HSA, which may be due to its ability to absorb other wavelengths or the presence of other substituents in the molecule. As described in our previous works [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], drug encapsulation can have a protective effect on changes in albumin chain packing (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and modifications of the secondary structure. Apart from our study, few authors have used CD in the analysis of polymer structure changes after preparation of nanoparticles or other materials [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe observed changes in the structure of HSA influenced by the presence of QBT at the molecular level, may not have a strong influence on the side effects generated in the \u003cem\u003ein vivo\u003c/em\u003e system, but further studies are required to unequivocally support this statement.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eThe research is a continuation of the attempt to encapsulation new quinobenzothiazine derivative into nanoparticles. Also, it allowed to analyze a quinobenzothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]thiazine) with potential anticancer activity using spectroscopic techniques and to attempt to encapsulate the compound in human albumin nanoparticles. Following the synthesis, particles with a diameter of 92.680 nm were obtained, and their diameter increased to 110.445 nm following the encapsulation process. Spectroscopic studies confirmed the presence of encapsulated QBT, which also allowed release studies to be performed. In order to ascertain the release kinetics mechanism, which is essential for the particles' medical applications, mathematical methods were implemented. In a biological environment, the nanocarriers' activity can be assessed through the release kinetics calculations and the graph of QBT release from the nanoparticles.\u003c/p\u003e\u003cp\u003eThe above results indicate the potential applicability of QBT-HSA-NPs in medicine. Due to the insufficiency of studies on QBT-containing nanoparticles, regardless of the promising results, further studies on albumin nanoparticles with QBT are required to further investigate the therapeutic potency of the structures formed. They can also form the basis for further preclinical and clinical studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Sources\u003c/h2\u003e \u003cp\u003eThis research was funded by the Medical University of Silesia, grant numbers: BNW-2-048/K/4/F, BNW-2-028/K/5/F, BNW-1-009/K/4/F, BNW-2-049/K/4/F, BNW-2-110/K/4/F.\u003c/p\u003e \u003cp\u003eACKNOWLEDGMENT\u003c/p\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKarolina Kulig \u0026ndash; experiment investigation performance and analyzed data, writing\u0026mdash;original draft, Patrycja Sarkowicz \u0026ndash; performed experiment, data analysis, contributed to the writing of the manuscript, Małgorzata Jeleń \u0026ndash; review and data discussion, Beata Morak-Młodawska \u0026ndash; review and data discussion, Magdalena Ziąbka \u0026ndash; SEM experiment investigation and performance, Aleksandra Owczarzy \u0026ndash; review and data discussion, Wojciech Rog\u0026oacute;ż \u0026ndash; review and data discussion, Małgorzata Maciążek-Jurczyk \u0026ndash; supervision, editing and linguistic assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhlawat J. 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ACS Appl Mater Interfaces 16(8):9868\u0026ndash;9879. https://doi.org/10.1021/acsami.3c18306\u003c/li\u003e\n\u003cli\u003eOwczarzy A, Rog\u0026oacute;ż W, Kulig K, Pożycka J, Zięba A, Maciążek-Jurczyk M (2023) Spectroscopic studies of quinobenzothiazine derivative in terms of the in vitro interaction with selected human plasma proteins: Part 2. Molecules 28(2):698. https://doi.org/10.3390/molecules28020698\u003c/li\u003e\n\u003cli\u003eCiepluch K, Biehl R, Bryszewska M, Arabski M (2020) Poly(propylene imine) dendrimers can bind to PEGylated albumin at PEG and albumin surface: Biophysical examination of a PEGylated platform to transport cationic dendritic nanoparticles. Biopolymers 111(9):e23386. https://doi.org/10.1002/bip.23386\u003c/li\u003e\n\u003cli\u003eMorozova OV, Pavlova ER, Bagrov DV, Barinov NA, Prusakov KA, Isaeva EI, Podgorsky VV, Basmanov DV, Klinov DV (2018) Protein nanoparticles with ligand-binding and enzymatic activities. Int J Nanomedicine 13:6637\u0026ndash;6646. https://doi.org/10.2147/IJN.S177627\u003c/li\u003e\n\u003cli\u003eVarytis P, Stefanou N, Christofi A, Papanikolaou N (2015) Strong circular dichroism of core-shell magnetoplasmonic nanoparticles. J Opt Soc Am B 32:1063\u0026ndash;1069. https://doi.org/10.1364/JOSAB.32.001063\u003c/li\u003e\n\u003cli\u003eTurner GA, Dunlap CE, Higgins AJ, Simpson GJ (2025) Dark-field absorbance circular dichroism of oriented chiral thin films. J Phys Chem Lett 30:1403\u0026ndash;1408. https://doi.org/10.1021/acs.jpclett.4c02984\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"colloid-and-polymer-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Colloid and Polymer Science](https://www.springer.com/journal/396) ","snPcode":"396","submissionUrl":"https://mc.manuscriptcentral.com/cps","title":"Colloid and Polymer Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"nanoparticles, albumin, phenothiazine derivative, spectroscopy","lastPublishedDoi":"10.21203/rs.3.rs-6749623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6749623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNanoparticles (NPs) provide a potential opportunity to reduce toxicity, optimize drug effects, and properly distribute drugs in the body and/or overcome multidrug resistance. Human serum albumin (HSA) is widely used as a drug carrier due to its biocompatibility and specific affinity to cancer cells. 6-Acetylaminobutyl-9-chloroquino[3,2-b]benzo[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]thiazine (QBT) is a tetracyclic, acetylaminobutyl phenothiazine derivative in which one of the benzene rings has been replaced by a quinoline. This compound has shown very promising \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e biological properties. The aim of this study was the spectroscopic analysis of QBT and the development of albumin nanoparticles (HSA-NPs) with encapsulated QBT (QBT-HSA-NPs). This study is a continuation of attempts to encapsulate phenothiazine derivatives in nanoparticles.\u003c/p\u003e \u003cp\u003eTo examine the spectroscopic properties of QBT, UV-Vis spectroscopy was applied. To investigate the properties of QBT to be encapsulated in HSA nanoparticles, the desolvation method was used. By using scanning electron microscopy (SEM), the size and shape of the nanoparticles were ascertained. The QBT release study was determined using the sampling and separation method and the mathematical drug release kinetics mechanism was estimated. Changes in the secondary structure of HSA were verified using circular dichroism (CD) spectropolarimetry.\u003c/p\u003e \u003cp\u003eQBT has an ability to absorb radiation in the UV-Vis range. The encapsulation efficiency was 97.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%, confirming that QBT can be encapsulated in HSA nanoparticles. SEM examination showed smooth nanoparticles of their size of 101.445\u0026thinsp;\u0026plusmn;\u0026thinsp;9.907 nm for QBT-HSA-NPs and 92.680\u0026thinsp;\u0026plusmn;\u0026thinsp;12.797 nm for HSA-NPs. QBT released according to the zero-order mechanism, via QBT diffusion and HSA swelling. The presence of QBT in nanoparticles partially protected the secondary structure of HSA. The observed changes in the structure of native HSA, influenced by the presence of QBT at the molecular level, may not have a strong influence on the side effects generated in the \u003cem\u003ein vivo\u003c/em\u003e system. Despite reports on albumin nanoparticles and QBT, no one has published studies on QBT encapsulation in nanoparticles to date.\u003c/p\u003e","manuscriptTitle":"Spectroscopic analysis of human serum albumin nanoparticles with encapsulated phenothiazine derivative (6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine) – continuation studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 10:51:46","doi":"10.21203/rs.3.rs-6749623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-23T09:54:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T10:40:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198229388292241380121207096879861245330","date":"2025-10-02T17:05:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287147373259382516861534245488327864895","date":"2025-10-01T04:43:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-29T19:13:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280804613786759051494850359962164980362","date":"2025-06-29T14:33:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195936903205664969607206889629998968017","date":"2025-06-24T08:04:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-24T07:55:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-02T23:58:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-02T23:57:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Colloid and Polymer Science","date":"2025-05-26T10:00:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"colloid-and-polymer-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Colloid and Polymer Science](https://www.springer.com/journal/396) ","snPcode":"396","submissionUrl":"https://mc.manuscriptcentral.com/cps","title":"Colloid and Polymer Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b60f93d-971a-4acc-9228-1b986a3b4d0d","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T08:54:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 10:51:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6749623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6749623","identity":"rs-6749623","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}