{"paper_id":"04761c7b-89f0-4f43-aaf6-b4bd45762b05","body_text":"Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy | 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 Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy Daniel González-García, Olga Tapia, Carmen Évora, Patricia García-García, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3977241/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jun, 2024 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. In this study, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG2000 were produced by single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs are successfully freeze-dried using 2.5 and 5% trehalose for long-term storage. The LPHNPs produced by MF and SSN increase 1.8–3.2 fold GapmeR cell uptake, respectively. They also endosomally escape in approximately 80%. The developed LPHNPs will be useful for targeting gene therapies. lipid-polymeric hybrid nanoparticles microfluidics gene therapy nanoparticles stability endosomal escape Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Gene therapy is a promising therapeutic option to treat a wide range of diseases by silencing over-producing proteins and genetically modifying and repairing cell functions that may affect disease conditions [ 1 ]. In the field of oligonucleotide-based therapies, GapmeRs are an emerging class of molecules that can knock down a target gene through post-transcriptional gene silencing [ 2 ]. Despite the great potential of GapmeRs, there are certain biopharmaceutical problems such as degradation risk, difficulties diffusing through biological membranes, and uncontrolled biodistribution due to their lack of specificity, low stability, and poor cellular internalization [ 3 ]. To overcome these issues, many formulation advances have been made over the past 30 years, including its encapsulation into non-viral delivery vectors [ 4 ]. Due to their minimal cytotoxicity and capacity to offer high oligonucleotide stability in the blood, lipid-polymeric hybrid nanoparticles (LPHNPs) have been shown to be highly successful for drug administration [ 5 ]. They comprise two essential components: a polymer core to encapsulate the drug and a lipid monolayer enveloping the core. The polymeric core provides stability and physical integrity during storage. In addition, the lipid shell serves as a molecular blockade, decreasing the leakage of enclosed drugs and shielding the core from deterioration by blocking water infiltration. The lipids also promote the cellular uptake and can be functionalized with cellular markers to increase specificity and reduce rapid clearance from blood circulation. These systems combine the advantages of lipid and polymeric nanoparticles (NPs), resulting in enhanced stability, biocompatibility, and cellular uptake [ 5 , 6 ]. However, the industrial development of LPHNPs is slowed by the lack of adequate production technology. Consequently, their clinical implementation progresses slower than conventional drug delivery systems. This is primarily due to the limitations associated with bulk preparation techniques. Single-step nanoprecipitation (SSN) is a widely used method for the preparation of LPHNPs because it is simple, rapid and economical [ 7 ]. However, this kind of production leads to challenges related to scaling up production, low reproducibility across batches, and the wide size distribution observed in the resulting NPs [ 8 ]. A recent advance in the production of NPs is microfluidic technology (MF). Microfluidics involves the laminar mixing of small volumes of solutions within milliseconds, allowing for the tuning of NPs properties such as size, surface charge, and encapsulation efficiency (EE) by changing concentrations of precursor reagents, the flow rate ratio (FRR), defined as the ratio between the flow rate of aqueous and organic solutions, and the total flow rate (TFR), defined as the sum of aqueous and organic flow rates [ 9 , 10 ]. Compared to conventional bulk techniques, the MF offers advantages in terms of standardization, certification, reproducibility and the facility to scale up production. Scalability in the production of oligonucleotide nanocarriers at an affordable cost is another important challenge to overcome for their widespread clinical use. It is important to point out that designing an effective nanocarrier to mitigate the issues associated with GapmeRs is a challenging task. The biological system's microenvironment, which is meant to be treated, has the ability to alter the properties of the nanocarrier. First of all, the GapmeRs can be complexed with cationic materials including protamine, DC-cholesterol, and chitosan to increase the yield of oligonucleotide encapsulation and enable their core localization. DC-cholesterol has been widely employed as a transfection agent [ 11 , 12 ] while low-molecular-weight protamine sulfate can shield oligonucleotides from biological degradation and promote their cellular penetration [ 13 , 14 ]. Because of its endosomolytic and oligonucleotide complexation capabilities, chitosan has also been used [ 15 ]. Size and zeta potential (ZP) must be carefully optimized in the preparation of NPs. Size plays a crucial role in cellular uptake, with smaller NPs exhibiting higher uptake than their larger counterparts. Size also affects drug release kinetics and biodistribution. ZP determines cellular uptake, biodistribution, and interactions within biological environments [ 16 ]. It has been observed that the cationic charge of NPs is strongly correlated with increased cellular uptake but is also associated with greater complement activation compared to negatively or neutrally charged particles [ 17 , 18 ]. Consequently, the use of ionizable lipids, such as cholesterol derivatives with tertiary amino groups, is an interesting strategy. These lipids are neutral at physiological pH to minimize toxicity and they can become protonated in the acidic endosome pH after their cellular uptake. This protonation facilitates membrane fusion or disruption, endosomal escape, and the release of cargo into the cytosol [ 19 ]. The 3-[N-(N,N - dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) offers the advantages of providing greater stability to lipid membranes and lower cytotoxicity as it is a molecule derived from the natural component cholesterol [ 12 ]. The utilization of pegylated lipids such as DOPE-mPEG 2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000) also enhances the stability of formulations and prolongs the circulation times of these systems. Despite the characteristics of these lipids, the use of DC-Chol and DOPE-mPEG 2000 in combination has only been widely used in the preparation of liposomes. In this study, the possible synergistic effect of both lipids on the properties of LPHNPs was explored. Taking into account the previously mentioned information, the main objective of this work is the encapsulation of a GampeR associated with a complexing agent in LPHNPs formulated with PLGA, DC-Chol, and DOPE-mPEG2000 using two different techniques: a convencional single-step nanoprecipitation method and microfluidic technology. A comparative analysis will be conducted to determine the formulation and technique that will enable the development of a better gene therapy platform in terms of smaller size and polydispersity index (PdI), positive ZP, EE, yield production, stability, cellular uptake, and lysosomal escape. 2. Materials and methods 2.1. Materials Poly (D,L-lactide-co-glycolide) (PLGA, Resomer® RG 502, Mr MW 7,000–17,000) was provided by Evonik (Germany), soy L-α-phosphatidylcholine (lecithin) and DC-Cholesterol (3β-[N-(N’,N’-dimethilaminoethane)-carbamoyl-cholesterol hydrochloride) were obtained from Avanti Polar Lipids (USA). DOPE-mPEG 2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) was provided by Nanosoft Polymers (USA). Chitosan hydrochloride (CHT, Protasan® UPCL-113, Mr MW: 50,000-150,000, DA: 82.5%) was purchased from Novamatrix (Norway). Protamine sulfate (Mr MW: 5,000–10,000) was purchased from Sigma-Aldrich (USA), GapmeR (MW 4928) and a 5’ fluorescently labelled GapmeR (MW 5465.5) with FAM was provided by Integrated DNA Technologies (USA). 2.2. GapmeR condensation Three cationic products (protamine, DC-Chol, and chitosan) were chosen to evaluate their ability to form non-covalent complexes with the oligonucleotide and improve its encapsulation. Protamine sulfate capacity to interact with GapmeR was assessed by electrophoresis as described in previous works of the research group [ 13 ]. Total condensation of the GapmeR occurred at a GapmeR:protamine mass ratio of 1:1. In the present study, to provide an excess of positive charges, the mass ratio used for LPHNPs preparation was 1:2.5. The charge ratio was estimated considering an average molecular weight of 7.5 kDa (Mr MW: 5,000–10,000) and an isoelectric point (pI) > 10 for protamine [ 20 , 21 ]. Consequently, to have the same charge ratio for GapmeR:DC-Chol or GapmeR:CHT, an estimation of the mass ratio was carried out using an average molecular weight for CHT of 100,000 (Mr MW: 50,000–150,000) and pKa 6.5 [ 22 ], and a molecular weight of DC-Chol of 537.3 and pKa 7.8 (D Pozzi et al, 2013). In addition, the degree of ionization of the different products in pure water (pH 7) and at pH 5.8 corresponding to air-equilibrated water [ 23 , 24 ] was also taken into account. 2.3. Development of the lipid-polymeric hybrid nanoparticles (LPHNPs), formulation and elaboration process. The LPHNPs are prepared by two different methods. One of them is a modification of the one-step nanoprecipitation method and another is the microfluidic technique. Regardless of the production method used, the formulations developed comprise an internal aqueous phase (fixed at 25 µl) containing the non-covalent complex of condensing agent and GapmeR, an organic phase (polymer solution) and an external aqueous phase. The organic phase consists of PLGA solutions in acetonitrile (1 ml) at a concentration range of 0.75–5 mg/ml. The outer aqueous phase is a 4% ethanol-aqueous solution of lipids in different proportions. All phases are filtrated through 0.45 µm syringe filters. The mass ratio of lipid/polymer used to prepare and optimize the nanoparticle formulations is in the range of 15–50%. The ratios of DOPE-mPEG 2000 /DC-Chol + lecithin are fixed at 1:3. Lecithin/DC-Chol ratios are tested at 0:3, 1:2, and 2:1. 2.3.1. Single-step nanoprecipitation (SSN). Briefly, 25 µl of the condensed-GapmeR aqueous solution are added to 1 ml of polymer solution in acetonitrile and mixed promptly by vortex. This mixture is immediately poured into 6 ml of hydro-alcoholic solution prepared with different amounts of lecithin, DOPE-mPEG 2000 , and DC-Cholesterol. The suspension is kept for 2.5 hours in an extraction hood at room temperature (RT) under magnetic stirring to allow acetonitrile evaporation. 2.3.2. Microfluidics (MF). The amounts of the lipids are dissolved in 3 ml of the hydro-alcoholic solution and loaded in 1.5 ml fractions into two syringes positioned on a syringe pump (KD Scientific, model 569, USA). This aqueous phase is pumped to the micromixerchip (Part N° 3200401, Dolomite Microfluidics, UK) at a flow rate range of 0.6-3 ml/min using a syringe pump (KD Scientific, model 569, USA). At the same time, 1 ml of the already mixed organic phase with the internal aqueous phase is also injected at a flow rate range of 0.2-1 ml/min using an automatic syringe pump (Mitos-Duo-XS-Pump, Dolomite Microfluidics, UK). Both phases get into the microfluidic chip through a ferule with an integrated filter (Part number 3200245, Dolomite Microfluidics) and are mixed for 5 min inside the chip. Afterwards, the resulting LPHNPs dispersion was collected in a beaker and kept under mild magnetic stirring for 2.5 hours for organic solvent evaporation. For elaboration process optimization by microfluidics, additional variables like Total Flow Rate (TFR) (the sum of aqueous and organic flow rates) in the system and the aqueous to organic Flow Rate Ratio (FRR) were examined. 2.4. LPHNPs Characterization For the characterization of the different formulations, the LPHNP suspensions were concentrated by ultrafiltration using Amicon® Ultra 100 KDa MWCO filters (Millipore, USA) at 10,000 rpm for 10 minutes and were resuspended to a final volume of 0.4 ml. 2.4.1. Physicochemical properties of LPHNPs The LPHNPs were characterized in terms of average hydrodynamic diameter, polydispersity index (PdI) and zeta potential (ZP) using a Zetasizer Nano-ZS (Malvern Instruments, UK). These characteristics were determined in the freshly prepared LPHNPs suspensions and after the concentration and resuspension processes. All measurements were performed in triplicate after an appropriate Milli-Q water dilution. 2.4.2. Transmission electron microscopy analysis Transmission electron microscopy (TEM) was utilized to examine the morphology and structure of LPHNPs. This analysis is also useful to validate the particle size previously determined via dynamic light scattering (DLS). 10 µl of each concentrated sample were deposited onto copper grids and coated with a carbon membrane. Subsequently, they were stained with a 1% (w/v) uranyl acetate solution for 2 minutes. Finally, the samples were examined at 120 kV using a JEOL microscope (JEM 2010, Japan) and images were captured with a Gatan Orius TM camera (USA). 2.4.3. Production yield The LPHNP suspensions were frozen at -80°C and subsequently freeze-dried. After this process, the content was weighed on a precision balance, and the production yield was calculated as follows: $$Production yield \\left(\\%\\right)=\\frac{mg of LHPNPs obtained after freeze-drying}{theorical mg \\left(sum of all reagent weights\\right)}\\times 100$$ 2.4.4. Oligonucleotide encapsulation efficiency and release Oligonucleotide encapsulation efficiency (EE) and release profile were evaluated using fluorescently labelled GapmeR (GapmeR-FAM). LPHNP batches were prepared with a 1 µg GapmeR loading, concentrated as described above and the filtrate containing the non-encapsulated GapmeR-FAM was measured using a plate reader at 485/528 nm. Furthermore, GapmeR release assays were carried out by incubating the concentrated LPHNPs suspension (350 µl) in 10% of fetal bovine serum (FBS, Lonza, Spain) phosphate buffers (prepared with DEPC water) to a final volume of 700 µl, at pH 7.4 and at pH 5.5 and at 37°C. In addition, the release assay was also carried out at pH 7.4 for 2 hours, followed by a medium change to pH 5.5, to simulate the different pHs in the LPHNPs distribution pathway after administration. At different times, the samples were ultrafiltered (100 kDa Amicon®Ultra filters, 10,000 rpm for 10 min), the GapmeR-FAM released into the filtrate solution was quantified as previously described [ 25 ], and LPHNPs were resuspended in fresh media. The assays were carried out in triplicate. 2.4.5. LPHNPs stability assay 2.4.5.1. Stability during storage The storage short-term colloidal stability of LPHNPs was tested at 4°C in water, 0.9% NaCl, and trehalose at 1.25%, 2.5%, and 10%. The LPHNP batches were suspended in the different media (final volume of 0.4 ml) and after 24 hours, LPHNPs size, PdI, and ZP were evaluated by DLS. For long-term preservation, each batch of LPHNPs (final volume of 3 ml) containing different proportions of trehalose (1.25, 2.5, 5, 7.5, and 10%) as cryoprotectant was frozen at -80°C and freeze-dried. Then, LPHNPs were resuspended in 1 ml of MilliQ water and characterized in terms of size, PdI, and ZP. 2.4.5.2. Stability in simulated biological media For this purpose, LPHNP stability was evaluated in 10% FBS in phosphate buffer saline (PBS) at pH 7.4 and pH 5.5. Additionally, LPHNP stability in a 4.5% aqueous solution of bovine serum albumin (BSA, Sigma-Aldrich, Spain) was studied. For this, concentrated LPHNPs were diluted tenfold in the different media and kept at 37°C under orbital shaking at 300 rpm. At different time points (0, 2, 6, 24 h), 100 µl of sample were withdrawn, and the physicochemical characteristics of LPHNPs were evaluated by DLS. 2.4.6. LPHNPs cell uptake and evaluation of lysosomal escape To assess LPHNPs uptake and evaluate the lysosomal escape, murine myoblasts (C2C12 cells) were seeded on sterile round glass coverslips in 12-well plates in complete DMEM (Dulbecco’s modified Eagle’s medium + 10% FBS, 1% peniciline/streptomicine, and 1% L-glutamine) at a density of 5x10 4 cells/well. When cells reached 60% confluence, they were incubated for 2 hours with 50 nM LysoTracker® Red DND-99 (Invitrogen, USA) in complete DMEM. After that period, the medium was replaced by LPHNP loaded with GapmeR-FAM suspended in complete DMEM at 2.4 mg/ml for 2 hours. Then, cells were washed twice with DPBS (Dulbecco’s Buffer Phosphate, Gibco, Thermo Fisher USA). A solution of free GampeR-FAM prepared with the same amount of GapmeR encapsulated in the LPHNPs was used as a control. Finally, the samples were mounted with ProLong Gold Antifade Mountant with DAPI and visualized by Leica SP8 confocal microscopy (Germany). The obtained images were analysed using image analysis software (ImageJ, v1.52, National Institute of Health, USA). 2.5. Statistical analysis The results are presented as the mean value ± SD. SPSS software (IBM SPSS Statistics v. 26) was used to conduct a one-way ANOVA, followed by Dunnett's post hoc test to compare the different groups with a control group. Statistically significant differences were considered at p < 0.05. 3. Results 3.1. GapmeR condensation For a 16-mer oligonucleotide, the GapmeR:protamine mass ratio of 1:2.5 can be expressed as a charge ratio of 1:2.3, regardless of the pH of pure water (pH 7) or air-equilibrated water (pH 5.8) because protamines are very basic peptides (IP > 10 [ 20 ]). Taking into account the molecular weight and degree of ionization in pure water and in air-equilibrate water of the DC-Chol and CHT, a mass ratio of GapmeR:DC-Chol of 1:5.4 and GapmeR:CHT of 1:1 was set to provide an excess amount of condensing agent to ensure the neutralization of the negative charges of the GapmeR. 3.2. LPHNP physicochemical and morphological characterization More than 30 different formulations were tested by SSN and MF but discarded due to their high size (> 200 nm) and/or PDI (> 0.3) or reproducibility issues. LPHNPs elaborated with a lipid/polymer ratio of > 20%, and regardless of both the PLGA amount used (1.25–5 mg) and the lecithin/DC-Chol ratio (1:2 or 2:1), have a size < 200 nm but a PdI > 0.3 and a negative ZP. However, for a lipid/polymer ratio of 15% using 1.25 mg of PLGA, nanoparticles with a suitable size (< 150 nm) and PdI < 0.3 were obtained, but with high ZP inter-batch variability. Consequently, lecithin was discarded from the LPHNPs composition, which also simplifies the formulations. Table 1 summarizes the physicochemical properties of LPHNP batches (n ≥ 3) of 27 formulations made by SSN without lecithin in their composition. The results show that LPHNPs made with 2.5–5 mg/ml PLGA are bigger than the ones with 1.25–2.5 mg/ml. This increase in LPHNPs size with polymer concentration was also described by other authors [ 26 ]. Regarding the lipid/polymer ratio, Table 1 shows that a ratio < 25% produces adequate size (140–180 nm) and PdI values (0.2–0.25) but negative ZP. To get a positive surface charge that could enhance cellular uptake and promote stability by electrostatic repulsion [ 27 ], the lipid/polymer ratio has to be increased to the 25–50% range. However, to avoid excess lipids that may not bind properly to the polymeric core surface [ 7 ], the lipid/polymer ratio was fixed at 25%. Considering these results, the LPHNPs with 1.25 mg of PLGA and a lipid/polymer mass ratio of 25% (size 149.9 ± 18.07 nm, PdI 0.23 ± 0.02, ZP 29.34 ± 2.44 mV) were employed to conduct further experiments. Table 1 Physicochemical characteristics (average size, PdI and ZP) of LPHNPs developed by single-step nanoprecipitation (SSN) using DOPE-PEG and DC-Chol (ratio 1:3) with different complexing agents: protamine sulfate (Prot), DC-Cholesterol (DC-Chol), and chitosan (CHT). PLGA (mg) Lipid/polymer mass ratio (%) Complexant Prot DC-Chol CHT 1.25–2.5 < 25 Size 140–180 nm, PdI ≤ 0.25 and negative ZP 25–50 Size 130–170 nm, PdI ≤ 0.25 and positive ZP 2.5-5 < 25 Size 170–180 nm, PdI ≤ 0.2 and neutral or positive ZP 25–50 Size 170–180 nm, PdI ≤ 0.25 and positive ZP In order to have analogous physicochemical features with microfluidics, several parameters, such as FRR, TFR, polymer mass and complexing agent were taken into account. Although the FRR was tested in the range of 3–10, it was set to 3 because at FRR > 3 an increase in PdI was observed (data not shown) caused by a shortening diffusion length that could affect the nucleation rate [ 28 ]. Table 2 sums up the physicochemical characteristics of LPHNPs (n ≥ 3) from 36 formulations and it reflects that PLGA amounts higher than 1.25 mg and/or TFR levels higher than 1.5 ml/min produce chip clogging by agglomeration. This could be due to the increase in organic phase viscosity which reduce organic solvent diffusion to the aqueous phase, but also to the intricate geometries of the chips that generate pressure resistance impacting the performance of the device [ 29 ]. As a result, all the formulations tested with TFR > 1 ml/min and PLGA mass > 1.25 mg have a size > 200 nm and PdI > 0.3. As indicated in Table 2 , LPHNPs formulated with TFR 1–1.5 ml/min do not meet the aforementioned requirements. However, the reduction in TFR down to 0.8 ml/min and the increase in lipid/polymer ratio up to 25% allowed production of positively charged LPHNPs (ZP 32.25 ± 1.36 mV), size of 179.8 ± 6.3 and a PdI of 0.24 ± 0.01. Table 2 Physicochemical characteristics (average size, PdI and ZP) of LPHNPs developed by MF using DOPE-PEG and DC-Chol (ratio 1:3) with different complexing agents: protamine sulfate (Prot), DC-Cholesterol (DC-Chol), and chitosan (CHT). TFR = total flow rate. PLGA (mg) Lipid/polymer mass ratio (%) TFR (ml/min) Complexant Prot DC-Chol CHT 0.75–1.25 < 25 0.8–1.5 Fluctuating ZP 1.6–3.2 Clogging of the chip 25–50 0.8 Size 140–180 nm and PdI ≤ 0.25 Size 140–180 nm and PdI ≤ 0.2 Size 160–180 and PdI ≤ 0.25 Positive ZP > 20 mV 1-1.5 Size > 200 nm and PdI > 0.3 Size 180–200 nm and PdI > 0.3 Size > 250 nm and PdI > 0.4 Positive ZP > 20 mV 1.6–3.2 Clogging of the chip 1.5–2.5 < 25–50 0.8–3.2 Figures 1 A and 1 B present the images from LPHNPs produced by SSN and MF, respectively. Analysis of Fig. 1 A confirms the presence of a homogeneous distribution in which the LPHNPs appear spherical throughout the sample. The pictures confirm the structure of the LPHNPs, formed by a polymeric core of PLGA surrounded by a lipid coating, whose components seem strongly interconnected with each other. LPHNPs obtained by MF (Fig. 1 B) are as uniform as the ones made by SNN with quite similar morphological characteristics. 3.3. Production yield, encapsulation efficiency, and release of GapmeR The production yield assessed by freeze-drying was 78% ± 3% in the case of SSN, while in MF yield increased to 86% ± 5%. These results show that both processes allow adequate production in relation to the mass of the reagents used. Regarding the encapsulation efficiency, the values obtained for LPHNPs elaborated by SSN and MF technique with protamine were 90% ± 4% and 91% ± 7%, respectively. The encapsulation efficiency using DC-Chol (SSN EE 46.9% ± 7.4 and MF EE 45.6% ± 3.26) or CHT (SSN EE 54.3% ± 5.5 and MF EE 58.1% ± 1.3) was much lower than using protamine. In view of this finding, protamine was chosen as the condensing agent to carry out this study. Concerning the GapmeR release from GapmeR:protamine-loaded LPHNPs (Fig. 2 A), the profiles are characterized by a high burst effect followed by a slower release rate and the total oligonucleotide dose was delivered in 24 hours, regardless of pH or production technique. To simulate a biodistribution process after LPHNP administration (blood and cell environment), a release assay consisting of a 2 h incubation at pH 7.4 followed by a 22-hour period at pH 5.5, was conducted. The release profiles observed in these conditions are practically superimposed on the pH 7.4 profiles (Fig. 2 B). 3.4. Stability assays Native LPHNP formulations optimized for both methods were not stable at 4°C after 6 hours (data not shown). To improve the storage of LPHNPs, they were resuspended in NaCl 0.9% to isotonize the suspension for potential future intravenous administration. However, the colloidal suspension was not stable at 4°C for 24 hours (Fig. 3 , A-B). Probably the greater ionic strength imparted by the 0.9% NaCl induces the diffuse double layer of the NPs to compress with the consequent aggregation due to the attractive forces (van der Waals) becoming greater than the electrostatic repulsive ones [ 30 ]. Alternatively, two concentrations of trehalose were used: 2.5 and 10%. At high trehalose concentrations, MF LPHNPs increase their size and PdI as opposed to SSN LPHNPs. However, a low trehalose concentration maintains size and PdI for both developed formulations (Fig. 3 , A-B). In view of these results, the long-term preservation of LPHNPs by freeze-drying was studied using trehalose in the range of 1.25–10% as cryoprotectant. After freeze-drying, LPHNPs were resuspended in 1 ml of MilliQ water by slight manual agitation and characterized in size, PdI and ZP. Highest concentrations of trehalose (7.5 and 10%) did not maintain the LPHNP characteristics after freeze-drying; sizes were greater than 1 µm and PdI > 0.9 (data not shown). This negative effect may be due to the high concentration and viscosity of the colloidal suspension which prevents the freezing of the total available water. This bound water leads to the formation of an amorphous, crystalline, or combined amorphous-crystalline phase [ 31 ]. Consequently, the lyophilization process failed and the LPHNPs were not resuspended properly. According to the results shown in Fig. 3 C-D, 1.25% of trehalose was insufficient for LPHNPs cryopreservation, while 2.5% and 5% maintained the characteristics of native LPHNPs after lyophilization, and they were easily resuspended. LPHNPs interaction with the biological environment is a very important event to consider in the production of theragnostic nanoformulations [ 32 , 33 ]. After administration, LPHNPs are immediately coated by proteins [ 34 ], but they are also exposed to pH changes. Most pH's tissues are similar to plasma pH; however, in endosomes, pH can drop to 5.5 [ 35 ]. In the present work, stability of LPHNPs incubated in phosphate buffer with 10% FBS at pH 7.4 and 5.5 was analysed at 37°C. Regardless of pH conditions, LPHNPs developed by both SSN and MF and incubated in 10% FBS maintained size, PdI and ZP during the first 6 hours. After 24 hours, the size increases 3–4 fold compared to the original value (Fig. 4 A), and PdI goes up to 7 (data not shown). This effect was less pronounced in LPHNPs studied at two pHs (preincubating at pH 7.4 for 2 hours). As albumin is the major protein in serum (3.4–5.4 g/dl), LPHNP stability was also tested by incubating LPHNPs in a 4.5% BSA aqueous solution at pH 7.4. There are no significant variations in size (Fig. 4 B), PdI or ZP (data not shown). Therefore, LPHNPs are stable for 24 hours. 3.5. Cellular uptake and lysosomal escape LPHNPs loaded with 5´-FAM-labeled GapmeR murine myoblasts (C2C12) uptake was assessed by confocal microscopy (Fig. 5 A-C). Densitometric analysis to quantify the total amount of FAM signal per cell revealed that LPHNPs prepared by SSN or MF were internalized 3.2 and 1.8 times higher than control cells (treated with non-encapsulated GapmeR), respectively (Fig. 5 D). Furthermore, co-staining using the lysosomal biomarker Red-DND-99 revealed that 84% of the GampeRs internalized by NPs had overcome the endosomal/lysosomal barrier, which represents a 1.2-fold increase compared to control cells (Fig. 5 E). 4. Discussion In the present work, we take advantage of the LPHNP characteristics and the two production techniques, conventional SSN and MF, to make a platform for GapmeR delivery. Several variables were evaluated to obtain NPs with suitable physicochemical characteristics. Regarding the lipid mixture composition, PEGylated lipids provide steric stabilization of the NP suspension during manufacturing and storage. PEGylation also prolongs circulation time and prevents uptake by the mononuclear phagocyte system [ 36 ], but has a downstream negative effect by decreasing cellular uptake. In this work, to achieve faster dePEGylation after administration, DOPE-mPEG 2000 was chosen instead of the more widely used DSPE-PEG because of its greater dissociation rate from NP surface conferred by its unsaturated chain [ 37 ]. Similarly to cholesterol, DC-Chol strongly interacts with polymer, imparting stability to the lipid shell. The positive charge of DC-Chol improves cellular uptake and at the same time acidic pH facilitates endosomal escape [ 38 ]. Additionally, it would improve NPs stability by electrostatic repulsion during storage. The effect of lipid/polymer mass ratio was tested in the range of 15–50%. At low lipid/polymer mass ratio (< 25%), NPs with neutral or negative Z-potential by SSN (Table 1 ) and flluctuating Z-potential or even a clogging of the chip by MF (Table 2 ) were produced, which indicate insuficient amount of lipids to complete core coating. The NP characteristics were not affected by the lipid/polymer ratio in the range of 25–50%. Thus, to prevent the formation of micelles or liposomes from the lipids not incorporated into the NP surface, the lipid/polymer mass ratio was set at 25%. As expected, an increase in particle size was observed when increasing PLGA concentration. Concentrations higher than 1.25 mg/mL were excessively viscous for the preparation of NPs by MF, producing chip clogging due to the low solvent diffusion rate. Aditional variables have to be considered for the NPs production by MF. TFR and FRR are the most mentioned parameters that influence the NPs size and PdI. An increase in both TFR and FRR leads to a decrease in size and PdI of lipid, polymeric and lipid-polymeric NPs. However, the micromixer design plays an important role in optimizing the process. Comparison of our results with others previously reported is difficult due to the wide variety of microchips used, which to some extent act as a limiting factor in the TFR and FRR that can be used. It has been reported that increases in FRR between 3 and 10 fold, depending on the geometry of the micromixer (homemade custom-made or commercial) and evidently the composition of the phases, lead to notable reductions in particle size [ 39 – 42 ]. However, the same authors also point out that the FRR has a limit above which further increases have no effect or a negative effect on size and/or PdI [ 39 – 41 ]. With respect to the TFR, some authors also show that the size reduction only occurs up to a certain limit beyond which the effect is reversed [ 43 ]. The micromixer chip used in the present study is a commercially available hydrophilic glass device designed to efficiently mix fluids. It combines the hydrodynamic focusing flow (HFF) at the inlet followed by 12 mixing stages consisting of series of alternate paths with different internal cross section (125 µm x 350 µm and 50 µm x 125 µm - depth x width) where a repeated fluid splitting and joining take place. HFF reduces the diffusion length of the organic solvent by compressing the central organic phase with the aqueous phase injected into the two symmetrical side channels. Like other authors,[ 39 – 41 ] the best results were obtained with a FFR of 3 because at higher ratio (tested from 3 to 10) NPs with PdI greater than the maximum established as optimal were obtained. The maximum TFR tolerated by this chip is 5 ml/min to avoid internal overpressure. However, at values greater than 1.5 ml/min (tested from 0.8 to 4 ml/min) the chip is clogging. In the range of 0.8 to 1.5 ml/min, an increase in TFR has a negative effect, leading to NPS with greater size and PdI (Table 2 ), the same as the behavior mentioned by Li et al. [ 43 ]. In any case, LPHNPs with optimal size (150–180 nm), PdI (0.23–0.24) and positive Z-potential (29–32 mV), were obtained by both conventional SSN and MF. It has to be highlighted that it was possible by using the same components and in the same proportion (1.25 mg/ml PLGA, lipid/polymer mas ratio of 25% and DOPE-mPEG 2000 at 25% molar ratio of total lipid) and setting FRR 3:1 and TFR of 0.8 ml/min for MF. The TEM images show NPs with a size and PdI consistent with the results obtained by DLS and similar structure which is compatible with that of a polymeric core surronded by a lipid shell. One of the strategies used to improve oligonucleotides encapsulation in LPHNPs is to form, through electrostatic and hydrophobic interactions, less water-soluble larger-size neutral complexes. The three condensing agents evaluated in the present work could also, improve the endosomal escape. Low molecular weight protamine is one of the most used condensing agents and with less toxicity. In fact, protamines act physiologically as condensers and DNA stabilizers in spermatozoa. However, changes in complexation efficiency have been reported between different protamines due to variations in amino acid composition and therefore in their conformation [ 44 ]. It is known that chitosan is also capable of complexing and condensing DNA molecules. The binding efficiency depends on the deacetylation degree and the presence of certain nucleotide sequences [ 45 ]. DC-Chol also forms multilaminar condensates, leaving the oligonucleotide chains confined between the lipid bilayers [ 46 ]. In our case, greater encapsulation efficiency with protamine, distantly followed by chitosan (56%) and DC-Cholesterol (46%) was obtained, regardless of the preparation technique (Table 1 and 2 ). In all cases, an excess of net charge was available for GapmeR complexation, therefore, the differences in encapsulation performances should be due to the own condensing agent structure and stability of the formed complex. Acording to the results, protamine sulfate was selected as a condensing agent for subsequent assays. The stability of NPs during storage plays an important role for translating to the clinic. Despite the steric and electrostatic stabilization (ZP 29–32 mV) provided by DOPE-mPEG 2000 and DC-Chol respectively, the NP suspensions did not maintain their physicochemical characteristics beyond 6 hours. On the contrary, they were effectively stabilized for the short and long storage using trehalose. NP suspensions in 2.5% trehalose were stable for at least 24 h at 4ºC, time enough for administration. NPs were also easily reconstituted after freeze-drying using trehalose in the range of 2.5-5% as cryoprotectant, providing suitable storage conditions for long-term use. Trehalose has also been recommended as a cryoprotectant for nanostructured solid lipid nanoparticles at concentrations in the range of 3.75–12.5% [ 47 ] and at 1–10% for polymeric particles [ 48 ]. However, in this study, concentrations higher than 7.5% do not work. Unfortunately, we have not found previous references to lyophilization of lipid-polymeric nanoparticles similar to those prepared by us. Consequently, the discrepancies with the above authors could be related with the different NP structure and composition but also to the NPs concentration to be freeze-dried. The physicochemical properties of nanoparticles, size, shape, surface charge and surface chemistry influence the efficiency of cellular uptake [ 49 , 50 ]. Upon contact with biological fluids or the culture media where they are going to be tested, NPs can change their physicochemical characteristics, which will affect their circulation time, distribution, release profile, interaction with target cells and endosomal escape. These changes are mainly due to the protein corona formation but also to the different pHs during intracellular trafficking. NPs with small sizes (30–50 nm) have greater cell penetration capacity and greater ability to escape from the mononuclear phagocyte system [ 51 ] and positive surface charges enhance the interaction with cells [ 52 ]. The usual mechanism of cellular uptake of NPs is by endocytosis. According to several authors NPs with a size lower than 200 nm preferentially use the clathrin pathway [ 53 , 54 ]. Endocytic vesicules fuse with the early endosomes (pH 6–7) which mature to late endosomes (pH 5.5-6) and finally to lysosomes (pH 4.5-5). Additionally, NPs can be exocyted throught recycling and exosomal exocytosis systems in any of this trafficking phases [ 55 ]. To exert their therapeutic action, oligonucleotides or oligonucleotide-NPs have to escape from the endosomal system and be released intact in the cytosol of the cell. Consecuently, the behavior of the NPs was evaluated in PBS pH 7.4 with both 4.5% BSA (Fig. 4 B) and 10% FBS, but also at pH 5.5 and at pH 7.4 for 2h followed by incubation at pH 5.5 in PBS with 10% FBS to simulate the intracellular trafficking (Fig. 4 A). The LPHNPs were stable for at least 24 hours in 4.5% BSA while the stability was reduced to 6 hours in 10% FBS. Regardless of the pH of the medium, after 24 h, the NPs increased in size, a sign of aggregation. NP aggregation could be produced by the exchange and/or the removal of the lipidic shell and by the adsorption of proteins at the NP surface. Due to the ionizable cationic DC-Chol (pKa 7.8) in the NP shell, the initial positive Z-potential (+ 29 mV) decreases to near neutrality (1–2 mV) after incubation in PBS (pH 7.4) with either BSA or FBS. The surfactant effect of BSA would keep the LPHNPs in suspension, while the complex composition of FBS with electrolytes and other substances could facilitate the loss of the lipid shell, increasing the formation of protein corona and agglomeration of the NPs. Similar behavior, after incubation in different biological fluids, in terms of decrease in Z potential and stability has been reported with PLGA NPs with a cationic polymer shell [ 56 ]. The GapmeR release profile was also affected by the pH of the medium. Specifically, a great burst release was observed at pH 5.5 (ca. 75%). At acidic pH DC-Chol is completely protonated and the formation of the protein corona is mainly through hydrophobic interactions and hydrogen bonds. At pH 7.4 some electrostatic interactions are possible, leading to the formation of a more compact protein corona that reduces the GapmeR release rate. However, the release was not modified when LPHNPs were previously incubated at pH 7.4 and then at pH 5.5, probably because this pH change does not affect the initial structure of the already formed protein corona. To evaluate the LPHNPS celular uptake efficiency the same dose of naked Gapmer was taken as a reference because as it previously reported single-stranded and relatively small oligonucleotides, uncharged and/or hydrophobic at high concentration can be cell uptaken and escape endosome without the invervention of any carrier [ 57 ]. Compared with the naked GapmeR, both celular uptake and endosomal escape were more efficient with LPHNPs (Fig. 5 ). Although unexpected high naked GapmeR endosomal escape was observed, combining both cell uptake and endosomal escape lead to an effective GapmeR delivery in the cytosol of approximately 2.5–4 fold higher with LPHNPs than with the naked GapmeR. The less cell uptake efficiency of LPHNPs prepared by MF technique compared with LPHNPs by SSN is difficult to explain since their physicochemical characteristics, stability and release profiles were similar. Even though the formulation components were the same, their disposition or location in the final formulation could not be, especially in the lipid shell. The production of NPs with a relatively complex structure like LPHNPs using such an efficient mixing system could trigger variations in the nucleation and coalescence process, which would lead to a different composition than expected. A higher PEGylation and lower amout of DC-Chol at the NPs surface could reduce the cellular uptake of the elaborated NPs. Ottonelli et al. [ 39 ] reported NPs with the similar physicochemical characteristics but different compositions depending on the preparation method (MF and SSN). 5. Conclusion We have described the production of LPHNPs with PLGA, DC-Chol and DOPE-mPEG 2000 by SSN and MF. The physicochemical properties were found to be sensitive to composition, polymer concentration, lipid/polymer ratio and MF parameters such as TFR and FFR, while the EE was affected by the complexing agent. The GapmeR-protamine-loaded LPHNPs demonstrated good stability in simulated biological conditions and suitability for long-term stability by freeze-drying. In addition, LPHNPs increase cell uptake of GapmeR and allow its endosomal escape in high rates. These findings provide a demonstration of the utility of the developed LPHNPs as highly effective oligonucleotide delivery vectors. Declarations Funding This work was supported by MCIN/AEI/10.13039/501100011033/FEDER, UE, (Grant numbers PID2021-127493OB-C21 and PID2021-126820OB-I00). Competing interests The authors have non-financial interests to disclose. Author Contributions Daniel González-García was involved in the conceptualization, methodology, investigation, data curation and original draft writing. Olga Tapia was involved in the methodology, investigation, funding acquisition and project administration. Carmen Évora was involved in the conceptualization, visualization, review and editing, funding acquisition, project administration and supervisión. Patricia García-García was involved in the conceptualization, methodology, investigation, formal analysis, original draft writing and editing. Araceli Delgado was involved in the conceptualization, visualization, original draft writing and editing, project administration, funding acquisition and supervision. All authors read and approved the final manuscript Data Availability The datasets generated during the current study are available from the corresponding author on reasonable request. Ethics approval Not applicable. References Hosseinkhani H et al. Gene Therapy for Regenerative Medicine . Pharmaceutics, 2023. 15(3). Fazil MH, et al. GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells. Sci Rep. 2016;6:37721. Gagliardi M, Ashizawa AT. The Challenges and Strategies of Antisense Oligonucleotide Drug Delivery . Biomedicines, 2021. 9(4). Wahane A et al. Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy . Molecules, 2020. 25(12). Merz L, et al. Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles. Eur J Pharm Biopharm. 2017;112:45–50. Mukherjee A, et al. Lipid-polymer hybrid nanoparticles as a next-generation drug delivery platform: state of the art, emerging technologies, and perspectives. Int J Nanomed. 2019;14:1937–52. Shah S, Famta P, Raghuvanshi RS, Singh SB, Srivastava S. Lipid polymer hybrid nanocarriers: Insights into synthesis aspects, characterization, release mechanisms, surface functionalization and potential implications. Colloids Interface Sci Commun. 2020;46:100570. Sivadasan D et al. Polymeric Lipid Hybrid Nanoparticles (PLNs) as Emerging Drug Delivery Platform-A Comprehensive Review of Their Properties, Preparation Methods, and Therapeutic Applications . Pharmaceutics, 2021. 13(8). Zhang HY, Sun J, Han R, Yang S, Teng Z. Microfluidics for nano-drug delivery systems: From fundamentals to industrialization. Acta Pharm Sin B. 2023;13:3277–99. Yanar F et al. Continuous-Flow Production of Liposomes with a Millireactor under Varying Fluidic Conditions . Pharmaceutics, 2020. 12(11). Zhang Y, et al. DC-Chol/DOPE cationic liposomes: a comparative study of the influence factors on plasmid pDNA and siRNA gene delivery. Int J Pharm. 2010;390(2):198–207. Li SG, Son X, Sorgi K, Hofland F, Huang H. DC-Chol lipid system in gene transfer. J Control Release. 1996;39:373–81. Garcia-Garcia P, et al. Tailor-made oligonucleotide-loaded lipid-polymer nanosystems designed for bone gene therapy. Drug Deliv Transl Res. 2021;11(2):598–607. Jarzebska NT et al. Protamine-Based Strategies for RNA Transfection . Pharmaceutics, 2021. 13(6). Ahmad A, Khan JM, Haque S. Strategies in the design of endosomolytic agents for facilitating endosomal escape in nanoparticles. Biochimie. 2019;160:61–75. Augustine RH, Primavera A, Wilson R, Thakor RJ, Kevadiya AS. Cellular uptake and retention of nanoparticles: Insights on particle properties and interaction with cellular components. Mater Today Commun. 2020;25:101692. Behzadi S, et al. Cellular uptake of nanoparticles: journey inside the cell. Chem Soc Rev. 2017;46(14):4218–44. Awashra M, Mlynarz P. The toxicity of nanoparticles and their interaction with cells: an in vitro metabolomic perspective. Nanoscale Adv. 2023;5(10):2674–723. Han X, et al. An ionizable lipid toolbox for RNA delivery. Nat Commun. 2021;12(1):7233. Ando TY, Suzuki M. Protamines. Isolation, characterization, structure and function. Mol Biol Biochem Biophys. 1973;12:1–114. Pugsley MK. Protamine , in Meyler's Side Effects of Drugs A. J.K., Editor. 2016. p. 1032–1034. Domard A. pH and c.d. measurements on a fully deacetylated chitosan: application to CuII—polymer interactions. Int J Biol Macromol. 1987;9:98–104. Willliam DW. Performance tests for the measurement of pH with glass electrodes in low ionic strength solutions including natural waters. Anal Chem. 1985;57(13):2567–70. Deleebeeck L et al. Unified pH Measurements of Ethanol, Methanol, and Acetonitrile, and Their Mixtures with Water . Sens (Basel), 2021. 21(11). Garcia-Garcia P, et al. Nanoparticle-mediated selective Sfrp-1 silencing enhances bone density in osteoporotic mice. J Nanobiotechnol. 2022;20(1):462. Dao TPT, To NTH, Ho VV, Nguyen TH, Dang TA. A new formulation of curcumin using poly (lactic-co-glycolic acid)—polyethylene glycol diblock copolymer as carrier material. Adv Nat Sci: Nanosci Nanotechnol. 2014;5:035013. Jeon S et al. Surface Charge-Dependent Cellular Uptake of Polystyrene Nanoparticles . Nanomaterials (Basel), 2018. 8(12). Bottaro E et al. Analysis of the Diffusion Process by pH Indicator in Microfluidic Chips for Liposome Production . Micromachines (Basel), 2017. 8(7). Gimondi S, et al. Microfluidic Devices: A Tool for Nanoparticle Synthesis and Performance Evaluation. ACS Nano. 2023;17(15):14205–28. Edwards SA, Williams DR. Double layers and interparticle forces in colloid science and biology: analytic results for the effect of ionic dispersion forces. Phys Rev Lett. 2004;92(24):248303. Abdelwahed W, et al. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev. 2006;58(15):1688–713. Falahati M, et al. A health concern regarding the protein corona, aggregation and disaggregation. Biochim Biophys Acta Gen Subj. 2019;1863(5):971–91. Caracciolo G, Farokhzad OC, Mahmoudi M. Biological Identity of Nanoparticles In Vivo: Clinical Implications of the Protein Corona. Trends Biotechnol, 2017. 35(3): p. 257–264. Pederzoli F et al. Protein corona and nanoparticles: how can we investigate on? . Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2017. 9(6). Hu Y, et al. Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability. Acta Biomater. 2015;28:149–59. Suk JS et al. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev, 2016. 99(Pt A): p. 28–51. Zhu X, et al. Surface De-PEGylation Controls Nanoparticle-Mediated siRNA Delivery In Vitro and In Vivo. Theranostics. 2017;7(7):1990–2002. Cardarelli F, et al. Cholesterol-dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO living cells. Mol Pharm. 2012;9(2):334–40. Ottonelli I et al. Microfluidic Technology for the Production of Hybrid Nanomedicines . Pharmaceutics, 2021. 13(9). Santhanes D, et al. Microfluidic formulation of lipid/polymer hybrid nanoparticles for plasmid DNA (pDNA) delivery. Int J Pharm. 2022;627:122223. Anderluzzi GP. Y, Microfluidic Manufacture of Solid Lipid Nanoparticles: A Case Study on Tristearin-Based Systems . Drug Deliv Lett, 2020. 10. Tahir N, et al. Microfluidic fabrication and characterization of Sorafenib-loaded lipid-polymer hybrid nanoparticles for controlled drug delivery. Int J Pharm. 2020;581:119275. Li Y et al. Synthesis of Polymer-Lipid Nanoparticles by Microfluidic Focusing for siRNA Delivery . Molecules, 2016. 21(10). Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther. 1997;4(9):961–8. Yang Y, et al. Binding efficacy and kinetics of chitosan with DNA duplex: The effects of deacetylation degree and nucleotide sequences. Carbohydr Polym. 2017;169:451–7. Wu CL, Chen W, Lin HL, Jeng TL. Self-Assembled Structure of the Binary Complex of DNA with Cationic Lipid. Macromolecules. 2004;37:4974–80. Rouco H et al. A Traffic Light System to Maximize Carbohydrate Cryoprotectants' Effectivity in Nanostructured Lipid Carriers' Lyophilization . Pharmaceutics, 2021. 13(9). Wong CY, Al-Salami H, Dass CR. Lyophilisation Improves Bioactivity and Stability of Insulin-Loaded Polymeric-Oligonucleotide Nanoparticles for Diabetes Treatment. Volume 21. AAPS PharmSciTech; 2020. p. 108. 3. Salatin S, Maleki Dizaj S, Yari A, Khosroushahi. Effect of the surface modification, size, and shape on cellular uptake of nanoparticles. Cell Biol Int. 2015;39(8):881–90. Treuel L, et al. Physicochemical characterization of nanoparticles and their behavior in the biological environment. Phys Chem Chem Phys. 2014;16(29):15053–67. Choi J, et al. Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophages. J Appl Toxicol. 2009;29(1):52–60. Kelf TA, et al. Non-specific cellular uptake of surface-functionalized quantum dots. Nanotechnology. 2010;21(28):285105. Rejman J, et al. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J. 2004;377(Pt 1):159–69. Sadhukha T, Prabha S. Encapsulation in nanoparticles improves anti-cancer efficacy of carboplatin. AAPS PharmSciTech. 2014;15(4):1029–38. Patel S, et al. Brief update on endocytosis of nanomedicines. Adv Drug Deliv Rev. 2019;144:90–111. Oliveira CL, et al. Characterization of polymeric nanoparticles for intravenous delivery: Focus on stability. Colloids Surf B Biointerfaces. 2017;150:326–33. Liang XH, et al. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat Biotechnol. 2016;34(8):875–80. Supplementary Files Graphicalabstract.png Cite Share Download PDF Status: Published Journal Publication published 12 Jun, 2024 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Major Revisions Needed 28 Mar, 2024 Reviewers agreed at journal 21 Feb, 2024 Reviewers invited by journal 21 Feb, 2024 Editor assigned by journal 20 Feb, 2024 First submitted to journal 20 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-3977241\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":274385977,\"identity\":\"ff688db3-ea3e-48fd-913b-a4dfc689ad1c\",\"order_by\":0,\"name\":\"Daniel González-García\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad de la Laguna\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Daniel\",\"middleName\":\"\",\"lastName\":\"González-García\",\"suffix\":\"\"},{\"id\":274385978,\"identity\":\"b2af0e3a-59c4-4a57-8761-387a08d479f1\",\"order_by\":1,\"name\":\"Olga Tapia\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad de la Laguna\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Olga\",\"middleName\":\"\",\"lastName\":\"Tapia\",\"suffix\":\"\"},{\"id\":274385979,\"identity\":\"c18f4fee-f420-453f-9c0b-287b8c2d00e6\",\"order_by\":2,\"name\":\"Carmen Évora\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad de la Laguna\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Carmen\",\"middleName\":\"\",\"lastName\":\"Évora\",\"suffix\":\"\"},{\"id\":274385980,\"identity\":\"b4098d59-e695-432f-8c4e-7cc754af63ce\",\"order_by\":3,\"name\":\"Patricia García-García\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidad de la Laguna\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Patricia\",\"middleName\":\"\",\"lastName\":\"García-García\",\"suffix\":\"\"},{\"id\":274385981,\"identity\":\"cd0b90c3-af50-4f50-b427-e684183ff697\",\"order_by\":4,\"name\":\"Araceli Delgado\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYFCCBMYDDAzMDPwMzI1gBjFaGMAqJRsYG0jUYnCAWC387ckHDn6psZYzvpEI1FJhndjA3v4ArxaJM88SDsscSzc2A2s5k57YwHPGAL81N3IMDkuwHU7cBtLC2HY4sUEiB78O+Rv5Hw5L/DucuHkGSAuQ0SD/HL/DDG7kMBz8CDR8gwRISwPIFgb8DjM888zgMGNfurHEmYcNBxKAnmrjycGvRe548sOHP75ZywGD7uCDDzXWsv3sx/E7DASYeWCsBCBmI6geCBh/EKNqFIyCUTAKRi4AAFWKVAh6Y2LBAAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0000-0001-7045-6933\",\"institution\":\"Universidad de la Laguna\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Araceli\",\"middleName\":\"\",\"lastName\":\"Delgado\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-02-22 01:43:57\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3977241/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3977241/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s13346-024-01644-4\",\"type\":\"published\",\"date\":\"2024-06-13T00:34:10+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":51642620,\"identity\":\"a49565e9-57aa-4b76-8978-768d7e048296\",\"added_by\":\"auto\",\"created_at\":\"2024-02-26 13:26:48\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":293296,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eImages from LPHNPs made by A) conventional single-step nanoprecipitation (SSN) and B) microfluidics (MF).\\u003c/em\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/5cc4a1cc439cb40f3f2c08e8.png\"},{\"id\":51642297,\"identity\":\"87d39b82-7e66-48c8-bd4d-3e3cda6b105a\",\"added_by\":\"auto\",\"created_at\":\"2024-02-26 13:18:48\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":75013,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGapmeR release profile from LPHNPs (A) at different pHs, 7.4 and 5.5 (B) at pH = 7.4 for the first two hours and continued at pH = 5.5 until the end of the assay. Continuous line and dotted line represent LPHNPs developed by SSN and MF, respectively. 37˚C and 300 rpm, n = 3.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/f9ed74b61811eadcdab6f3c9.png\"},{\"id\":51642300,\"identity\":\"843c0a7f-f21d-40c7-a333-70839880992e\",\"added_by\":\"auto\",\"created_at\":\"2024-02-26 13:18:48\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":192159,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Sizes and (B) PdI of LPHNPs in different stabilizers: 0.9% NaCl, 2.5% trehalose and 10% trehalose at time 0 and 24 hours at 4°C. (C) Sizes and (D) PdI of LPHNPs dispersed in different concentrations of trehalose before and after being freeze-dried and resuspended in 1 ml of MilliQ water. Filled bars and striped bars represent LPHNPs developed by SSN and MF, respectively. Symbols denote statistically significant differences with native LPHNPs at time zero, prepared by SSN (#) and prepared by MF (*). n=3, p\\u0026lt;0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/576b4e3c188b67574e127028.png\"},{\"id\":51642299,\"identity\":\"7bc7b144-95a4-4a58-aaec-4e70be5dddd5\",\"added_by\":\"auto\",\"created_at\":\"2024-02-26 13:18:48\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":329530,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIncrease in LPHNPs size elaborated by SSN and MF (A) in 10% FBS at different pHs, 7.4, 5.5, and 7.4 during the first 2 hours and 5.5 during the rest of the assay (B) in 4.5% BSA at pH 7.4. Filled bars and striped bars represent LPHNPs developed by SSN and MF, respectively. Symbols denote statistically significant differences with native LPHNPs at time zero prepared by SSN (#) and prepared by MF (*). n=3, p\\u0026lt;0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/7f4b03d7278e96d61e1297b3.png\"},{\"id\":51642302,\"identity\":\"dee8076c-7cf2-4a6b-a1a9-2dc6d6222ac9\",\"added_by\":\"auto\",\"created_at\":\"2024-02-26 13:18:48\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":795841,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A-C) Confocal representative images (A) naked GapmeR-FAM; (B) LPHNPs loaded with GapmeR-FAM elaborated by SSN; (C) LPHNPs loaded with GapmeR-FAM elaborated by MF. (D) Densitometric analysis to quantify the internalization ratio was calculated with respect to the GapmeR-FAM control. (E) Lysosomal escape (%) of GapmeR control, LPHNPs by SSN and LPHNPs by MF. Nanoparticles are labelled in green, lysosomes are marked in red and cell nuclei are stained in blue. Scale bar: 25 µm; (**) denotes statistically significant differences with the GapmeR-FAM control. p\\u0026lt;0.05 (n = 3).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/9679603a5353785b857e0888.png\"},{\"id\":58321851,\"identity\":\"8379b938-226d-41e1-99ee-5fa35599ed39\",\"added_by\":\"auto\",\"created_at\":\"2024-06-14 00:34:20\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2449684,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/808b190c-2257-4f54-b1a2-dbdd01bc7d72.pdf\"},{\"id\":51642619,\"identity\":\"5e6a8820-4ec9-4734-9c77-07eed17aa6a5\",\"added_by\":\"auto\",\"created_at\":\"2024-02-26 13:26:48\",\"extension\":\"png\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":135619,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Graphicalabstract.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3977241/v1/6c1962f2bfef5ed73f372098.png\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eGene therapy is a promising therapeutic option to treat a wide range of diseases by silencing over-producing proteins and genetically modifying and repairing cell functions that may affect disease conditions [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. In the field of oligonucleotide-based therapies, GapmeRs are an emerging class of molecules that can knock down a target gene through post-transcriptional gene silencing [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Despite the great potential of GapmeRs, there are certain biopharmaceutical problems such as degradation risk, difficulties diffusing through biological membranes, and uncontrolled biodistribution due to their lack of specificity, low stability, and poor cellular internalization [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTo overcome these issues, many formulation advances have been made over the past 30 years, including its encapsulation into non-viral delivery vectors [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Due to their minimal cytotoxicity and capacity to offer high oligonucleotide stability in the blood, lipid-polymeric hybrid nanoparticles (LPHNPs) have been shown to be highly successful for drug administration [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. They comprise two essential components: a polymer core to encapsulate the drug and a lipid monolayer enveloping the core. The polymeric core provides stability and physical integrity during storage. In addition, the lipid shell serves as a molecular blockade, decreasing the leakage of enclosed drugs and shielding the core from deterioration by blocking water infiltration. The lipids also promote the cellular uptake and can be functionalized with cellular markers to increase specificity and reduce rapid clearance from blood circulation. These systems combine the advantages of lipid and polymeric nanoparticles (NPs), resulting in enhanced stability, biocompatibility, and cellular uptake [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eHowever, the industrial development of LPHNPs is slowed by the lack of adequate production technology. Consequently, their clinical implementation progresses slower than conventional drug delivery systems. This is primarily due to the limitations associated with bulk preparation techniques. Single-step nanoprecipitation (SSN) is a widely used method for the preparation of LPHNPs because it is simple, rapid and economical [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. However, this kind of production leads to challenges related to scaling up production, low reproducibility across batches, and the wide size distribution observed in the resulting NPs [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eA recent advance in the production of NPs is microfluidic technology (MF). Microfluidics involves the laminar mixing of small volumes of solutions within milliseconds, allowing for the tuning of NPs properties such as size, surface charge, and encapsulation efficiency (EE) by changing concentrations of precursor reagents, the flow rate ratio (FRR), defined as the ratio between the flow rate of aqueous and organic solutions, and the total flow rate (TFR), defined as the sum of aqueous and organic flow rates [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Compared to conventional bulk techniques, the MF offers advantages in terms of standardization, certification, reproducibility and the facility to scale up production. Scalability in the production of oligonucleotide nanocarriers at an affordable cost is another important challenge to overcome for their widespread clinical use.\\u003c/p\\u003e \\u003cp\\u003eIt is important to point out that designing an effective nanocarrier to mitigate the issues associated with GapmeRs is a challenging task. The biological system's microenvironment, which is meant to be treated, has the ability to alter the properties of the nanocarrier.\\u003c/p\\u003e \\u003cp\\u003eFirst of all, the GapmeRs can be complexed with cationic materials including protamine, DC-cholesterol, and chitosan to increase the yield of oligonucleotide encapsulation and enable their core localization. DC-cholesterol has been widely employed as a transfection agent [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e] while low-molecular-weight protamine sulfate can shield oligonucleotides from biological degradation and promote their cellular penetration [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Because of its endosomolytic and oligonucleotide complexation capabilities, chitosan has also been used [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eSize and zeta potential (ZP) must be carefully optimized in the preparation of NPs. Size plays a crucial role in cellular uptake, with smaller NPs exhibiting higher uptake than their larger counterparts. Size also affects drug release kinetics and biodistribution. ZP determines cellular uptake, biodistribution, and interactions within biological environments [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. It has been observed that the cationic charge of NPs is strongly correlated with increased cellular uptake but is also associated with greater complement activation compared to negatively or neutrally charged particles [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eConsequently, the use of ionizable lipids, such as cholesterol derivatives with tertiary amino groups, is an interesting strategy. These lipids are neutral at physiological pH to minimize toxicity and they can become protonated in the acidic endosome pH after their cellular uptake. This protonation facilitates membrane fusion or disruption, endosomal escape, and the release of cargo into the cytosol [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. The 3-[N-(N,N - dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) offers the advantages of providing greater stability to lipid membranes and lower cytotoxicity as it is a molecule derived from the natural component cholesterol [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. The utilization of pegylated lipids such as DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000) also enhances the stability of formulations and prolongs the circulation times of these systems. Despite the characteristics of these lipids, the use of DC-Chol and DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e in combination has only been widely used in the preparation of liposomes. In this study, the possible synergistic effect of both lipids on the properties of LPHNPs was explored.\\u003c/p\\u003e \\u003cp\\u003eTaking into account the previously mentioned information, the main objective of this work is the encapsulation of a GampeR associated with a complexing agent in LPHNPs formulated with PLGA, DC-Chol, and DOPE-mPEG2000 using two different techniques: a convencional single-step nanoprecipitation method and microfluidic technology. A comparative analysis will be conducted to determine the formulation and technique that will enable the development of a better gene therapy platform in terms of smaller size and polydispersity index (PdI), positive ZP, EE, yield production, stability, cellular uptake, and lysosomal escape.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Materials\\u003c/h2\\u003e \\u003cp\\u003ePoly (D,L-lactide-co-glycolide) (PLGA, Resomer\\u0026reg; RG 502, Mr MW 7,000\\u0026ndash;17,000) was provided by Evonik (Germany), soy L-α-phosphatidylcholine (lecithin) and DC-Cholesterol (3β-[N-(N\\u0026rsquo;,N\\u0026rsquo;-dimethilaminoethane)-carbamoyl-cholesterol hydrochloride) were obtained from Avanti Polar Lipids (USA). DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)) was provided by Nanosoft Polymers (USA). Chitosan hydrochloride (CHT, Protasan\\u0026reg; UPCL-113, Mr MW: 50,000-150,000, DA: 82.5%) was purchased from Novamatrix (Norway). Protamine sulfate (Mr MW: 5,000\\u0026ndash;10,000) was purchased from Sigma-Aldrich (USA), GapmeR (MW 4928) and a 5\\u0026rsquo; fluorescently labelled GapmeR (MW 5465.5) with FAM was provided by Integrated DNA Technologies (USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. GapmeR condensation\\u003c/h2\\u003e \\u003cp\\u003eThree cationic products (protamine, DC-Chol, and chitosan) were chosen to evaluate their ability to form non-covalent complexes with the oligonucleotide and improve its encapsulation.\\u003c/p\\u003e \\u003cp\\u003eProtamine sulfate capacity to interact with GapmeR was assessed by electrophoresis as described in previous works of the research group [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Total condensation of the GapmeR occurred at a GapmeR:protamine mass ratio of 1:1. In the present study, to provide an excess of positive charges, the mass ratio used for LPHNPs preparation was 1:2.5. The charge ratio was estimated considering an average molecular weight of 7.5 kDa (Mr MW: 5,000\\u0026ndash;10,000) and an isoelectric point (pI)\\u0026thinsp;\\u0026gt;\\u0026thinsp;10 for protamine [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Consequently, to have the same charge ratio for GapmeR:DC-Chol or GapmeR:CHT, an estimation of the mass ratio was carried out using an average molecular weight for CHT of 100,000 (Mr MW: 50,000\\u0026ndash;150,000) and pKa 6.5 [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e], and a molecular weight of DC-Chol of 537.3 and pKa 7.8 (D Pozzi et al, 2013). In addition, the degree of ionization of the different products in pure water (pH 7) and at pH 5.8 corresponding to air-equilibrated water [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] was also taken into account.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Development of the lipid-polymeric hybrid nanoparticles (LPHNPs), formulation and elaboration process.\\u003c/h2\\u003e \\u003cp\\u003eThe LPHNPs are prepared by two different methods. One of them is a modification of the one-step nanoprecipitation method and another is the microfluidic technique.\\u003c/p\\u003e \\u003cp\\u003eRegardless of the production method used, the formulations developed comprise an internal aqueous phase (fixed at 25 \\u0026micro;l) containing the non-covalent complex of condensing agent and GapmeR, an organic phase (polymer solution) and an external aqueous phase. The organic phase consists of PLGA solutions in acetonitrile (1 ml) at a concentration range of 0.75\\u0026ndash;5 mg/ml. The outer aqueous phase is a 4% ethanol-aqueous solution of lipids in different proportions. All phases are filtrated through 0.45 \\u0026micro;m syringe filters.\\u003c/p\\u003e \\u003cp\\u003eThe mass ratio of lipid/polymer used to prepare and optimize the nanoparticle formulations is in the range of 15\\u0026ndash;50%. The ratios of DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e/DC-Chol\\u0026thinsp;+\\u0026thinsp;lecithin are fixed at 1:3. Lecithin/DC-Chol ratios are tested at 0:3, 1:2, and 2:1.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1. Single-step nanoprecipitation (SSN).\\u003c/h2\\u003e \\u003cp\\u003eBriefly, 25 \\u0026micro;l of the condensed-GapmeR aqueous solution are added to 1 ml of polymer solution in acetonitrile and mixed promptly by vortex. This mixture is immediately poured into 6 ml of hydro-alcoholic solution prepared with different amounts of lecithin, DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e, and DC-Cholesterol. The suspension is kept for 2.5 hours in an extraction hood at room temperature (RT) under magnetic stirring to allow acetonitrile evaporation.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2. Microfluidics (MF).\\u003c/h2\\u003e \\u003cp\\u003eThe amounts of the lipids are dissolved in 3 ml of the hydro-alcoholic solution and loaded in 1.5 ml fractions into two syringes positioned on a syringe pump (KD Scientific, model 569, USA). This aqueous phase is pumped to the micromixerchip (Part N\\u0026deg; 3200401, Dolomite Microfluidics, UK) at a flow rate range of 0.6-3 ml/min using a syringe pump (KD Scientific, model 569, USA). At the same time, 1 ml of the already mixed organic phase with the internal aqueous phase is also injected at a flow rate range of 0.2-1 ml/min using an automatic syringe pump (Mitos-Duo-XS-Pump, Dolomite Microfluidics, UK). Both phases get into the microfluidic chip through a ferule with an integrated filter (Part number 3200245, Dolomite Microfluidics) and are mixed for 5 min inside the chip. Afterwards, the resulting LPHNPs dispersion was collected in a beaker and kept under mild magnetic stirring for 2.5 hours for organic solvent evaporation.\\u003c/p\\u003e \\u003cp\\u003eFor elaboration process optimization by microfluidics, additional variables like Total Flow Rate (TFR) (the sum of aqueous and organic flow rates) in the system and the aqueous to organic Flow Rate Ratio (FRR) were examined.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. LPHNPs Characterization\\u003c/h2\\u003e \\u003cp\\u003eFor the characterization of the different formulations, the LPHNP suspensions were concentrated by ultrafiltration using Amicon\\u0026reg; Ultra 100 KDa MWCO filters (Millipore, USA) at 10,000 rpm for 10 minutes and were resuspended to a final volume of 0.4 ml.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.1. Physicochemical properties of LPHNPs\\u003c/h2\\u003e \\u003cp\\u003eThe LPHNPs were characterized in terms of average hydrodynamic diameter, polydispersity index (PdI) and zeta potential (ZP) using a Zetasizer Nano-ZS (Malvern Instruments, UK). These characteristics were determined in the freshly prepared LPHNPs suspensions and after the concentration and resuspension processes. All measurements were performed in triplicate after an appropriate Milli-Q water dilution.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.2. Transmission electron microscopy analysis\\u003c/h2\\u003e \\u003cp\\u003eTransmission electron microscopy (TEM) was utilized to examine the morphology and structure of LPHNPs. This analysis is also useful to validate the particle size previously determined via dynamic light scattering (DLS). 10 \\u0026micro;l of each concentrated sample were deposited onto copper grids and coated with a carbon membrane. Subsequently, they were stained with a 1% (w/v) uranyl acetate solution for 2 minutes. Finally, the samples were examined at 120 kV using a JEOL microscope (JEM 2010, Japan) and images were captured with a Gatan Orius TM camera (USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.3. Production yield\\u003c/h2\\u003e \\u003cp\\u003eThe LPHNP suspensions were frozen at -80\\u0026deg;C and subsequently freeze-dried. After this process, the content was weighed on a precision balance, and the production yield was calculated as follows:\\u003cdiv id=\\\"Equa\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equa\\\" name=\\\"EquationSource\\\"\\u003e\\n$$Production yield \\\\left(\\\\%\\\\right)=\\\\frac{mg of LHPNPs obtained after freeze-drying}{theorical mg \\\\left(sum of all reagent weights\\\\right)}\\\\times 100$$\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.4. Oligonucleotide encapsulation efficiency and release\\u003c/h2\\u003e \\u003cp\\u003eOligonucleotide encapsulation efficiency (EE) and release profile were evaluated using fluorescently labelled GapmeR (GapmeR-FAM). LPHNP batches were prepared with a 1 \\u0026micro;g GapmeR loading, concentrated as described above and the filtrate containing the non-encapsulated GapmeR-FAM was measured using a plate reader at 485/528 nm. Furthermore, GapmeR release assays were carried out by incubating the concentrated LPHNPs suspension (350 \\u0026micro;l) in 10% of fetal bovine serum (FBS, Lonza, Spain) phosphate buffers (prepared with DEPC water) to a final volume of 700 \\u0026micro;l, at pH 7.4 and at pH 5.5 and at 37\\u0026deg;C. In addition, the release assay was also carried out at pH 7.4 for 2 hours, followed by a medium change to pH 5.5, to simulate the different pHs in the LPHNPs distribution pathway after administration. At different times, the samples were ultrafiltered (100 kDa Amicon\\u0026reg;Ultra filters, 10,000 rpm for 10 min), the GapmeR-FAM released into the filtrate solution was quantified as previously described [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], and LPHNPs were resuspended in fresh media. The assays were carried out in triplicate.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.5. LPHNPs stability assay\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e2.4.5.1. Stability during storage\\u003c/h2\\u003e \\u003cp\\u003eThe storage short-term colloidal stability of LPHNPs was tested at 4\\u0026deg;C in water, 0.9% NaCl, and trehalose at 1.25%, 2.5%, and 10%. The LPHNP batches were suspended in the different media (final volume of 0.4 ml) and after 24 hours, LPHNPs size, PdI, and ZP were evaluated by DLS. For long-term preservation, each batch of LPHNPs (final volume of 3 ml) containing different proportions of trehalose (1.25, 2.5, 5, 7.5, and 10%) as cryoprotectant was frozen at -80\\u0026deg;C and freeze-dried. Then, LPHNPs were resuspended in 1 ml of MilliQ water and characterized in terms of size, PdI, and ZP.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003e2.4.5.2. Stability in simulated biological media\\u003c/h2\\u003e \\u003cp\\u003eFor this purpose, LPHNP stability was evaluated in 10% FBS in phosphate buffer saline (PBS) at pH 7.4 and pH 5.5. Additionally, LPHNP stability in a 4.5% aqueous solution of bovine serum albumin (BSA, Sigma-Aldrich, Spain) was studied. For this, concentrated LPHNPs were diluted tenfold in the different media and kept at 37\\u0026deg;C under orbital shaking at 300 rpm. At different time points (0, 2, 6, 24 h), 100 \\u0026micro;l of sample were withdrawn, and the physicochemical characteristics of LPHNPs were evaluated by DLS.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.6. LPHNPs cell uptake and evaluation of lysosomal escape\\u003c/h2\\u003e \\u003cp\\u003eTo assess LPHNPs uptake and evaluate the lysosomal escape, murine myoblasts (C2C12 cells) were seeded on sterile round glass coverslips in 12-well plates in complete DMEM (Dulbecco\\u0026rsquo;s modified Eagle\\u0026rsquo;s medium\\u0026thinsp;+\\u0026thinsp;10% FBS, 1% peniciline/streptomicine, and 1% L-glutamine) at a density of 5x10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well. When cells reached 60% confluence, they were incubated for 2 hours with 50 nM LysoTracker\\u0026reg; Red DND-99 (Invitrogen, USA) in complete DMEM. After that period, the medium was replaced by LPHNP loaded with GapmeR-FAM suspended in complete DMEM at 2.4 mg/ml for 2 hours. Then, cells were washed twice with DPBS (Dulbecco\\u0026rsquo;s Buffer Phosphate, Gibco, Thermo Fisher USA). A solution of free GampeR-FAM prepared with the same amount of GapmeR encapsulated in the LPHNPs was used as a control. Finally, the samples were mounted with ProLong Gold Antifade Mountant with DAPI and visualized by Leica SP8 confocal microscopy (Germany). The obtained images were analysed using image analysis software (ImageJ, v1.52, National Institute of Health, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe results are presented as the mean value\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD. SPSS software (IBM SPSS Statistics v. 26) was used to conduct a one-way ANOVA, followed by Dunnett's post hoc test to compare the different groups with a control group. Statistically significant differences were considered at p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. GapmeR condensation\\u003c/h2\\u003e \\u003cp\\u003eFor a 16-mer oligonucleotide, the GapmeR:protamine mass ratio of 1:2.5 can be expressed as a charge ratio of 1:2.3, regardless of the pH of pure water (pH 7) or air-equilibrated water (pH 5.8) because protamines are very basic peptides (IP\\u0026thinsp;\\u0026gt;\\u0026thinsp;10 [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]). Taking into account the molecular weight and degree of ionization in pure water and in air-equilibrate water of the DC-Chol and CHT, a mass ratio of GapmeR:DC-Chol of 1:5.4 and GapmeR:CHT of 1:1 was set to provide an excess amount of condensing agent to ensure the neutralization of the negative charges of the GapmeR.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. LPHNP physicochemical and morphological characterization\\u003c/h2\\u003e \\u003cp\\u003eMore than 30 different formulations were tested by SSN and MF but discarded due to their high size (\\u0026gt;\\u0026thinsp;200 nm) and/or PDI (\\u0026gt;\\u0026thinsp;0.3) or reproducibility issues. LPHNPs elaborated with a lipid/polymer ratio of \\u0026gt;\\u0026thinsp;20%, and regardless of both the PLGA amount used (1.25\\u0026ndash;5 mg) and the lecithin/DC-Chol ratio (1:2 or 2:1), have a size\\u0026thinsp;\\u0026lt;\\u0026thinsp;200 nm but a PdI\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.3 and a negative ZP. However, for a lipid/polymer ratio of 15% using 1.25 mg of PLGA, nanoparticles with a suitable size (\\u0026lt;\\u0026thinsp;150 nm) and PdI\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.3 were obtained, but with high ZP inter-batch variability. Consequently, lecithin was discarded from the LPHNPs composition, which also simplifies the formulations.\\u003c/p\\u003e \\u003cp\\u003eTable\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e summarizes the physicochemical properties of LPHNP batches (n\\u0026thinsp;\\u0026ge;\\u0026thinsp;3) of 27 formulations made by SSN without lecithin in their composition. The results show that LPHNPs made with 2.5\\u0026ndash;5 mg/ml PLGA are bigger than the ones with 1.25\\u0026ndash;2.5 mg/ml. This increase in LPHNPs size with polymer concentration was also described by other authors [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Regarding the lipid/polymer ratio, Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e shows that a ratio\\u0026thinsp;\\u0026lt;\\u0026thinsp;25% produces adequate size (140\\u0026ndash;180 nm) and PdI values (0.2\\u0026ndash;0.25) but negative ZP. To get a positive surface charge that could enhance cellular uptake and promote stability by electrostatic repulsion [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e], the lipid/polymer ratio has to be increased to the 25\\u0026ndash;50% range. However, to avoid excess lipids that may not bind properly to the polymeric core surface [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], the lipid/polymer ratio was fixed at 25%. Considering these results, the LPHNPs with 1.25 mg of PLGA and a lipid/polymer mass ratio of 25% (size 149.9\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;18.07 nm, PdI 0.23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02, ZP 29.34\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.44 mV) were employed to conduct further experiments.\\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\\u003ePhysicochemical characteristics (average size, PdI and ZP) of LPHNPs developed by single-step nanoprecipitation (SSN) using DOPE-PEG and DC-Chol (ratio 1:3) with different complexing agents: protamine sulfate (Prot), DC-Cholesterol (DC-Chol), and chitosan (CHT).\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\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=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003ePLGA (mg)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eLipid/polymer mass ratio (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eComplexant\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eProt\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eDC-Chol\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eCHT\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e1.25\\u0026ndash;2.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eSize 140\\u0026ndash;180 nm, PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.25 and negative ZP\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e25\\u0026ndash;50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eSize 130\\u0026ndash;170 nm, PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.25 and positive ZP\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e2.5-5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eSize 170\\u0026ndash;180 nm, PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.2 and neutral or positive ZP\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e25\\u0026ndash;50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e \\u003cp\\u003eSize 170\\u0026ndash;180 nm, PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.25 and positive ZP\\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 order to have analogous physicochemical features with microfluidics, several parameters, such as FRR, TFR, polymer mass and complexing agent were taken into account. Although the FRR was tested in the range of 3\\u0026ndash;10, it was set to 3 because at FRR\\u0026thinsp;\\u0026gt;\\u0026thinsp;3 an increase in PdI was observed (data not shown) caused by a shortening diffusion length that could affect the nucleation rate [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e sums up the physicochemical characteristics of LPHNPs (n\\u0026thinsp;\\u0026ge;\\u0026thinsp;3) from 36 formulations and it reflects that PLGA amounts higher than 1.25 mg and/or TFR levels higher than 1.5 ml/min produce chip clogging by agglomeration. This could be due to the increase in organic phase viscosity which reduce organic solvent diffusion to the aqueous phase, but also to the intricate geometries of the chips that generate pressure resistance impacting the performance of the device [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. As a result, all the formulations tested with TFR\\u0026thinsp;\\u0026gt;\\u0026thinsp;1 ml/min and PLGA mass\\u0026thinsp;\\u0026gt;\\u0026thinsp;1.25 mg have a size\\u0026thinsp;\\u0026gt;\\u0026thinsp;200 nm and PdI\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.3. As indicated in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, LPHNPs formulated with TFR 1\\u0026ndash;1.5 ml/min do not meet the aforementioned requirements. However, the reduction in TFR down to 0.8 ml/min and the increase in lipid/polymer ratio up to 25% allowed production of positively charged LPHNPs (ZP 32.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.36 mV), size of 179.8\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.3 and a PdI of 0.24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01.\\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\\u003ePhysicochemical characteristics (average size, PdI and ZP) of LPHNPs developed by MF using DOPE-PEG and DC-Chol (ratio 1:3) with different complexing agents: protamine sulfate (Prot), DC-Cholesterol (DC-Chol), and chitosan (CHT). TFR\\u0026thinsp;=\\u0026thinsp;total flow rate.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"8\\\"\\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=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003ePLGA (mg)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eLipid/polymer mass ratio (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eTFR (ml/min)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"5\\\" nameend=\\\"c8\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eComplexant\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eProt\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c7\\\" namest=\\\"c6\\\"\\u003e \\u003cp\\u003eDC-Chol\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003eCHT\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"6\\\" rowspan=\\\"7\\\"\\u003e \\u003cp\\u003e0.75\\u0026ndash;1.25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.8\\u0026ndash;1.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"5\\\" nameend=\\\"c8\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eFluctuating ZP\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.6\\u0026ndash;3.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"5\\\" nameend=\\\"c8\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003eClogging of the chip\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"4\\\" rowspan=\\\"5\\\"\\u003e \\u003cp\\u003e25\\u0026ndash;50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e0.8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eSize 140\\u0026ndash;180 nm and PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c6\\\" namest=\\\"c5\\\"\\u003e \\u003cp\\u003eSize 140\\u0026ndash;180 nm and PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e \\u003cp\\u003eSize 160\\u0026ndash;180 and PdI\\u0026thinsp;\\u0026le;\\u0026thinsp;0.25\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"5\\\" nameend=\\\"c8\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003ePositive ZP\\u0026thinsp;\\u0026gt;\\u0026thinsp;20 mV\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e1-1.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eSize\\u0026thinsp;\\u0026gt;\\u0026thinsp;200 nm and PdI\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c6\\\" namest=\\\"c5\\\"\\u003e \\u003cp\\u003eSize 180\\u0026ndash;200 nm and PdI\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e \\u003cp\\u003eSize\\u0026thinsp;\\u0026gt;\\u0026thinsp;250 nm and PdI\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"5\\\" nameend=\\\"c8\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003ePositive ZP\\u0026thinsp;\\u0026gt;\\u0026thinsp;20 mV\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.6\\u0026ndash;3.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"5\\\" morerows=\\\"1\\\" nameend=\\\"c8\\\" namest=\\\"c4\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eClogging of the chip\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e1.5\\u0026ndash;2.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;25\\u0026ndash;50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.8\\u0026ndash;3.2\\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\\u003eFigures \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA and \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB present the images from LPHNPs produced by SSN and MF, respectively. Analysis of Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA confirms the presence of a homogeneous distribution in which the LPHNPs appear spherical throughout the sample. The pictures confirm the structure of the LPHNPs, formed by a polymeric core of PLGA surrounded by a lipid coating, whose components seem strongly interconnected with each other. LPHNPs obtained by MF (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB) are as uniform as the ones made by SNN with quite similar morphological characteristics.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. Production yield, encapsulation efficiency, and release of GapmeR\\u003c/h2\\u003e \\u003cp\\u003eThe production yield assessed by freeze-drying was 78% \\u0026plusmn; 3% in the case of SSN, while in MF yield increased to 86% \\u0026plusmn; 5%. These results show that both processes allow adequate production in relation to the mass of the reagents used.\\u003c/p\\u003e \\u003cp\\u003eRegarding the encapsulation efficiency, the values obtained for LPHNPs elaborated by SSN and MF technique with protamine were 90% \\u0026plusmn; 4% and 91% \\u0026plusmn; 7%, respectively. The encapsulation efficiency using DC-Chol (SSN EE 46.9% \\u0026plusmn; 7.4 and MF EE 45.6% \\u0026plusmn; 3.26) or CHT (SSN EE 54.3% \\u0026plusmn; 5.5 and MF EE 58.1% \\u0026plusmn; 1.3) was much lower than using protamine. In view of this finding, protamine was chosen as the condensing agent to carry out this study.\\u003c/p\\u003e \\u003cp\\u003eConcerning the GapmeR release from GapmeR:protamine-loaded LPHNPs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA), the profiles are characterized by a high burst effect followed by a slower release rate and the total oligonucleotide dose was delivered in 24 hours, regardless of pH or production technique. To simulate a biodistribution process after LPHNP administration (blood and cell environment), a release assay consisting of a 2 h incubation at pH 7.4 followed by a 22-hour period at pH 5.5, was conducted. The release profiles observed in these conditions are practically superimposed on the pH 7.4 profiles (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. Stability assays\\u003c/h2\\u003e \\u003cp\\u003eNative LPHNP formulations optimized for both methods were not stable at 4\\u0026deg;C after 6 hours (data not shown). To improve the storage of LPHNPs, they were resuspended in NaCl 0.9% to isotonize the suspension for potential future intravenous administration. However, the colloidal suspension was not stable at 4\\u0026deg;C for 24 hours (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, A-B). Probably the greater ionic strength imparted by the 0.9% NaCl induces the diffuse double layer of the NPs to compress with the consequent aggregation due to the attractive forces (van der Waals) becoming greater than the electrostatic repulsive ones [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Alternatively, two concentrations of trehalose were used: 2.5 and 10%. At high trehalose concentrations, MF LPHNPs increase their size and PdI as opposed to SSN LPHNPs. However, a low trehalose concentration maintains size and PdI for both developed formulations (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, A-B). In view of these results, the long-term preservation of LPHNPs by freeze-drying was studied using trehalose in the range of 1.25\\u0026ndash;10% as cryoprotectant. After freeze-drying, LPHNPs were resuspended in 1 ml of MilliQ water by slight manual agitation and characterized in size, PdI and ZP. Highest concentrations of trehalose (7.5 and 10%) did not maintain the LPHNP characteristics after freeze-drying; sizes were greater than 1 \\u0026micro;m and PdI\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.9 (data not shown). This negative effect may be due to the high concentration and viscosity of the colloidal suspension which prevents the freezing of the total available water. This bound water leads to the formation of an amorphous, crystalline, or combined amorphous-crystalline phase [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Consequently, the lyophilization process failed and the LPHNPs were not resuspended properly. According to the results shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC-D, 1.25% of trehalose was insufficient for LPHNPs cryopreservation, while 2.5% and 5% maintained the characteristics of native LPHNPs after lyophilization, and they were easily resuspended.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eLPHNPs interaction with the biological environment is a very important event to consider in the production of theragnostic nanoformulations [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. After administration, LPHNPs are immediately coated by proteins [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], but they are also exposed to pH changes. Most pH's tissues are similar to plasma pH; however, in endosomes, pH can drop to 5.5 [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn the present work, stability of LPHNPs incubated in phosphate buffer with 10% FBS at pH 7.4 and 5.5 was analysed at 37\\u0026deg;C. Regardless of pH conditions, LPHNPs developed by both SSN and MF and incubated in 10% FBS maintained size, PdI and ZP during the first 6 hours. After 24 hours, the size increases 3\\u0026ndash;4 fold compared to the original value (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA), and PdI goes up to 7 (data not shown). This effect was less pronounced in LPHNPs studied at two pHs (preincubating at pH 7.4 for 2 hours).\\u003c/p\\u003e \\u003cp\\u003eAs albumin is the major protein in serum (3.4\\u0026ndash;5.4 g/dl), LPHNP stability was also tested by incubating LPHNPs in a 4.5% BSA aqueous solution at pH 7.4. There are no significant variations in size (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB), PdI or ZP (data not shown). Therefore, LPHNPs are stable for 24 hours.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5. Cellular uptake and lysosomal escape\\u003c/h2\\u003e \\u003cp\\u003eLPHNPs loaded with 5\\u0026acute;-FAM-labeled GapmeR murine myoblasts (C2C12) uptake was assessed by confocal microscopy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA-C). Densitometric analysis to quantify the total amount of FAM signal per cell revealed that LPHNPs prepared by SSN or MF were internalized 3.2 and 1.8 times higher than control cells (treated with non-encapsulated GapmeR), respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). Furthermore, co-staining using the lysosomal biomarker Red-DND-99 revealed that 84% of the GampeRs internalized by NPs had overcome the endosomal/lysosomal barrier, which represents a 1.2-fold increase compared to control cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eIn the present work, we take advantage of the LPHNP characteristics and the two production techniques, conventional SSN and MF, to make a platform for GapmeR delivery. Several variables were evaluated to obtain NPs with suitable physicochemical characteristics.\\u003c/p\\u003e \\u003cp\\u003eRegarding the lipid mixture composition, PEGylated lipids provide steric stabilization of the NP suspension during manufacturing and storage. PEGylation also prolongs circulation time and prevents uptake by the mononuclear phagocyte system [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e], but has a downstream negative effect by decreasing cellular uptake. In this work, to achieve faster dePEGylation after administration, DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e was chosen instead of the more widely used DSPE-PEG because of its greater dissociation rate from NP surface conferred by its unsaturated chain [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Similarly to cholesterol, DC-Chol strongly interacts with polymer, imparting stability to the lipid shell. The positive charge of DC-Chol improves cellular uptake and at the same time acidic pH facilitates endosomal escape [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Additionally, it would improve NPs stability by electrostatic repulsion during storage. The effect of lipid/polymer mass ratio was tested in the range of 15\\u0026ndash;50%. At low lipid/polymer mass ratio (\\u0026lt;\\u0026thinsp;25%), NPs with neutral or negative Z-potential by SSN (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) and flluctuating Z-potential or even a clogging of the chip by MF (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e) were produced, which indicate insuficient amount of lipids to complete core coating. The NP characteristics were not affected by the lipid/polymer ratio in the range of 25\\u0026ndash;50%. Thus, to prevent the formation of micelles or liposomes from the lipids not incorporated into the NP surface, the lipid/polymer mass ratio was set at 25%. As expected, an increase in particle size was observed when increasing PLGA concentration. Concentrations higher than 1.25 mg/mL were excessively viscous for the preparation of NPs by MF, producing chip clogging due to the low solvent diffusion rate.\\u003c/p\\u003e \\u003cp\\u003eAditional variables have to be considered for the NPs production by MF. TFR and FRR are the most mentioned parameters that influence the NPs size and PdI. An increase in both TFR and FRR leads to a decrease in size and PdI of lipid, polymeric and lipid-polymeric NPs. However, the micromixer design plays an important role in optimizing the process. Comparison of our results with others previously reported is difficult due to the wide variety of microchips used, which to some extent act as a limiting factor in the TFR and FRR that can be used. It has been reported that increases in FRR between 3 and 10 fold, depending on the geometry of the micromixer (homemade custom-made or commercial) and evidently the composition of the phases, lead to notable reductions in particle size [\\u003cspan additionalcitationids=\\\"CR40 CR41\\\" citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. However, the same authors also point out that the FRR has a limit above which further increases have no effect or a negative effect on size and/or PdI [\\u003cspan additionalcitationids=\\\"CR40\\\" citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. With respect to the TFR, some authors also show that the size reduction only occurs up to a certain limit beyond which the effect is reversed [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe micromixer chip used in the present study is a commercially available hydrophilic glass device designed to efficiently mix fluids. It combines the hydrodynamic focusing flow (HFF) at the inlet followed by 12 mixing stages consisting of series of alternate paths with different internal cross section (125 \\u0026micro;m x 350 \\u0026micro;m and 50 \\u0026micro;m x 125 \\u0026micro;m - depth x width) where a repeated fluid splitting and joining take place. HFF reduces the diffusion length of the organic solvent by compressing the central organic phase with the aqueous phase injected into the two symmetrical side channels. Like other authors,[\\u003cspan additionalcitationids=\\\"CR40\\\" citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e] the best results were obtained with a FFR of 3 because at higher ratio (tested from 3 to 10) NPs with PdI greater than the maximum established as optimal were obtained. The maximum TFR tolerated by this chip is 5 ml/min to avoid internal overpressure. However, at values greater than 1.5 ml/min (tested from 0.8 to 4 ml/min) the chip is clogging. In the range of 0.8 to 1.5 ml/min, an increase in TFR has a negative effect, leading to NPS with greater size and PdI (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), the same as the behavior mentioned by Li et al. [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn any case, LPHNPs with optimal size (150\\u0026ndash;180 nm), PdI (0.23\\u0026ndash;0.24) and positive Z-potential (29\\u0026ndash;32 mV), were obtained by both conventional SSN and MF. It has to be highlighted that it was possible by using the same components and in the same proportion (1.25 mg/ml PLGA, lipid/polymer mas ratio of 25% and DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e at 25% molar ratio of total lipid) and setting FRR 3:1 and TFR of 0.8 ml/min for MF. The TEM images show NPs with a size and PdI consistent with the results obtained by DLS and similar structure which is compatible with that of a polymeric core surronded by a lipid shell.\\u003c/p\\u003e \\u003cp\\u003eOne of the strategies used to improve oligonucleotides encapsulation in LPHNPs is to form, through electrostatic and hydrophobic interactions, less water-soluble larger-size neutral complexes. The three condensing agents evaluated in the present work could also, improve the endosomal escape. Low molecular weight protamine is one of the most used condensing agents and with less toxicity. In fact, protamines act physiologically as condensers and DNA stabilizers in spermatozoa. However, changes in complexation efficiency have been reported between different protamines due to variations in amino acid composition and therefore in their conformation [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. It is known that chitosan is also capable of complexing and condensing DNA molecules. The binding efficiency depends on the deacetylation degree and the presence of certain nucleotide sequences [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. DC-Chol also forms multilaminar condensates, leaving the oligonucleotide chains confined between the lipid bilayers [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. In our case, greater encapsulation efficiency with protamine, distantly followed by chitosan (56%) and DC-Cholesterol (46%) was obtained, regardless of the preparation technique (Table \\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and \\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). In all cases, an excess of net charge was available for GapmeR complexation, therefore, the differences in encapsulation performances should be due to the own condensing agent structure and stability of the formed complex. Acording to the results, protamine sulfate was selected as a condensing agent for subsequent assays.\\u003c/p\\u003e \\u003cp\\u003eThe stability of NPs during storage plays an important role for translating to the clinic. Despite the steric and electrostatic stabilization (ZP 29\\u0026ndash;32 mV) provided by DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e and DC-Chol respectively, the NP suspensions did not maintain their physicochemical characteristics beyond 6 hours. On the contrary, they were effectively stabilized for the short and long storage using trehalose. NP suspensions in 2.5% trehalose were stable for at least 24 h at 4\\u0026ordm;C, time enough for administration. NPs were also easily reconstituted after freeze-drying using trehalose in the range of 2.5-5% as cryoprotectant, providing suitable storage conditions for long-term use. Trehalose has also been recommended as a cryoprotectant for nanostructured solid lipid nanoparticles at concentrations in the range of 3.75\\u0026ndash;12.5% [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e] and at 1\\u0026ndash;10% for polymeric particles [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. However, in this study, concentrations higher than 7.5% do not work. Unfortunately, we have not found previous references to lyophilization of lipid-polymeric nanoparticles similar to those prepared by us. Consequently, the discrepancies with the above authors could be related with the different NP structure and composition but also to the NPs concentration to be freeze-dried.\\u003c/p\\u003e \\u003cp\\u003eThe physicochemical properties of nanoparticles, size, shape, surface charge and surface chemistry influence the efficiency of cellular uptake [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. Upon contact with biological fluids or the culture media where they are going to be tested, NPs can change their physicochemical characteristics, which will affect their circulation time, distribution, release profile, interaction with target cells and endosomal escape. These changes are mainly due to the protein corona formation but also to the different pHs during intracellular trafficking. NPs with small sizes (30\\u0026ndash;50 nm) have greater cell penetration capacity and greater ability to escape from the mononuclear phagocyte system [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e] and positive surface charges enhance the interaction with cells [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. The usual mechanism of cellular uptake of NPs is by endocytosis. According to several authors NPs with a size lower than 200 nm preferentially use the clathrin pathway [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. Endocytic vesicules fuse with the early endosomes (pH 6\\u0026ndash;7) which mature to late endosomes (pH 5.5-6) and finally to lysosomes (pH 4.5-5). Additionally, NPs can be exocyted throught recycling and exosomal exocytosis systems in any of this trafficking phases [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. To exert their therapeutic action, oligonucleotides or oligonucleotide-NPs have to escape from the endosomal system and be released intact in the cytosol of the cell. Consecuently, the behavior of the NPs was evaluated in PBS pH 7.4 with both 4.5% BSA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB) and 10% FBS, but also at pH 5.5 and at pH 7.4 for 2h followed by incubation at pH 5.5 in PBS with 10% FBS to simulate the intracellular trafficking (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The LPHNPs were stable for at least 24 hours in 4.5% BSA while the stability was reduced to 6 hours in 10% FBS. Regardless of the pH of the medium, after 24 h, the NPs increased in size, a sign of aggregation. NP aggregation could be produced by the exchange and/or the removal of the lipidic shell and by the adsorption of proteins at the NP surface. Due to the ionizable cationic DC-Chol (pKa 7.8) in the NP shell, the initial positive Z-potential (+\\u0026thinsp;29 mV) decreases to near neutrality (1\\u0026ndash;2 mV) after incubation in PBS (pH 7.4) with either BSA or FBS. The surfactant effect of BSA would keep the LPHNPs in suspension, while the complex composition of FBS with electrolytes and other substances could facilitate the loss of the lipid shell, increasing the formation of protein corona and agglomeration of the NPs. Similar behavior, after incubation in different biological fluids, in terms of decrease in Z potential and stability has been reported with PLGA NPs with a cationic polymer shell [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe GapmeR release profile was also affected by the pH of the medium. Specifically, a great burst release was observed at pH 5.5 (ca. 75%). At acidic pH DC-Chol is completely protonated and the formation of the protein corona is mainly through hydrophobic interactions and hydrogen bonds. At pH 7.4 some electrostatic interactions are possible, leading to the formation of a more compact protein corona that reduces the GapmeR release rate. However, the release was not modified when LPHNPs were previously incubated at pH 7.4 and then at pH 5.5, probably because this pH change does not affect the initial structure of the already formed protein corona.\\u003c/p\\u003e \\u003cp\\u003eTo evaluate the LPHNPS celular uptake efficiency the same dose of naked Gapmer was taken as a reference because as it previously reported single-stranded and relatively small oligonucleotides, uncharged and/or hydrophobic at high concentration can be cell uptaken and escape endosome without the invervention of any carrier [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. Compared with the naked GapmeR, both celular uptake and endosomal escape were more efficient with LPHNPs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Although unexpected high naked GapmeR endosomal escape was observed, combining both cell uptake and endosomal escape lead to an effective GapmeR delivery in the cytosol of approximately 2.5\\u0026ndash;4 fold higher with LPHNPs than with the naked GapmeR. The less cell uptake efficiency of LPHNPs prepared by MF technique compared with LPHNPs by SSN is difficult to explain since their physicochemical characteristics, stability and release profiles were similar. Even though the formulation components were the same, their disposition or location in the final formulation could not be, especially in the lipid shell. The production of NPs with a relatively complex structure like LPHNPs using such an efficient mixing system could trigger variations in the nucleation and coalescence process, which would lead to a different composition than expected. A higher PEGylation and lower amout of DC-Chol at the NPs surface could reduce the cellular uptake of the elaborated NPs. Ottonelli et al. [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e] reported NPs with the similar physicochemical characteristics but different compositions depending on the preparation method (MF and SSN).\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eWe have described the production of LPHNPs with PLGA, DC-Chol and DOPE-mPEG\\u003csub\\u003e2000\\u003c/sub\\u003e by SSN and MF. The physicochemical properties were found to be sensitive to composition, polymer concentration, lipid/polymer ratio and MF parameters such as TFR and FFR, while the EE was affected by the complexing agent. The GapmeR-protamine-loaded LPHNPs demonstrated good stability in simulated biological conditions and suitability for long-term stability by freeze-drying. In addition, LPHNPs increase cell uptake of GapmeR and allow its endosomal escape in high rates. These findings provide a demonstration of the utility of the developed LPHNPs as highly effective oligonucleotide delivery vectors.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eFunding\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by MCIN/AEI/10.13039/501100011033/FEDER, UE, (Grant numbers PID2021-127493OB-C21 and PID2021-126820OB-I00).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCompeting interests\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have non-financial interests to disclose.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAuthor Contributions\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDaniel Gonz\\u0026aacute;lez-Garc\\u0026iacute;a was involved in the conceptualization, methodology, investigation, data curation and original draft writing. Olga Tapia was involved in the methodology, investigation, funding acquisition and project administration. Carmen \\u0026Eacute;vora was involved in the conceptualization, visualization, review and editing, funding acquisition, project administration and supervisi\\u0026oacute;n. Patricia Garc\\u0026iacute;a-Garc\\u0026iacute;a was involved in the conceptualization, methodology, investigation, formal analysis, original draft writing and editing. Araceli Delgado was involved in the conceptualization, visualization, original draft writing and editing, project administration, funding acquisition and supervision. All authors read and approved the final manuscript\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eData Availability\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eEthics approval\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eHosseinkhani H et al. \\u003cem\\u003eGene Therapy for Regenerative Medicine\\u003c/em\\u003e. Pharmaceutics, 2023. 15(3).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFazil MH, et al. GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells. Sci Rep. 2016;6:37721.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGagliardi M, Ashizawa AT. \\u003cem\\u003eThe Challenges and Strategies of Antisense Oligonucleotide Drug Delivery\\u003c/em\\u003e. Biomedicines, 2021. 9(4).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWahane A et al. \\u003cem\\u003eRole of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy\\u003c/em\\u003e. Molecules, 2020. 25(12).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMerz L, et al. Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles. Eur J Pharm Biopharm. 2017;112:45\\u0026ndash;50.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMukherjee A, et al. Lipid-polymer hybrid nanoparticles as a next-generation drug delivery platform: state of the art, emerging technologies, and perspectives. Int J Nanomed. 2019;14:1937\\u0026ndash;52.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eShah S, Famta P, Raghuvanshi RS, Singh SB, Srivastava S. Lipid polymer hybrid nanocarriers: Insights into synthesis aspects, characterization, release mechanisms, surface functionalization and potential implications. Colloids Interface Sci Commun. 2020;46:100570.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSivadasan D et al. \\u003cem\\u003ePolymeric Lipid Hybrid Nanoparticles (PLNs) as Emerging Drug Delivery Platform-A Comprehensive Review of Their Properties, Preparation Methods, and Therapeutic Applications\\u003c/em\\u003e. Pharmaceutics, 2021. 13(8).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang HY, Sun J, Han R, Yang S, Teng Z. Microfluidics for nano-drug delivery systems: From fundamentals to industrialization. Acta Pharm Sin B. 2023;13:3277\\u0026ndash;99.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYanar F et al. \\u003cem\\u003eContinuous-Flow Production of Liposomes with a Millireactor under Varying Fluidic Conditions\\u003c/em\\u003e. Pharmaceutics, 2020. 12(11).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang Y, et al. DC-Chol/DOPE cationic liposomes: a comparative study of the influence factors on plasmid pDNA and siRNA gene delivery. Int J Pharm. 2010;390(2):198\\u0026ndash;207.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi SG, Son X, Sorgi K, Hofland F, Huang H. DC-Chol lipid system in gene transfer. J Control Release. 1996;39:373\\u0026ndash;81.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGarcia-Garcia P, et al. Tailor-made oligonucleotide-loaded lipid-polymer nanosystems designed for bone gene therapy. Drug Deliv Transl Res. 2021;11(2):598\\u0026ndash;607.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJarzebska NT et al. \\u003cem\\u003eProtamine-Based Strategies for RNA Transfection\\u003c/em\\u003e. Pharmaceutics, 2021. 13(6).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAhmad A, Khan JM, Haque S. Strategies in the design of endosomolytic agents for facilitating endosomal escape in nanoparticles. Biochimie. 2019;160:61\\u0026ndash;75.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAugustine RH, Primavera A, Wilson R, Thakor RJ, Kevadiya AS. Cellular uptake and retention of nanoparticles: Insights on particle properties and interaction with cellular components. Mater Today Commun. 2020;25:101692.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBehzadi S, et al. Cellular uptake of nanoparticles: journey inside the cell. Chem Soc Rev. 2017;46(14):4218\\u0026ndash;44.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAwashra M, Mlynarz P. The toxicity of nanoparticles and their interaction with cells: an in vitro metabolomic perspective. Nanoscale Adv. 2023;5(10):2674\\u0026ndash;723.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHan X, et al. An ionizable lipid toolbox for RNA delivery. Nat Commun. 2021;12(1):7233.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAndo TY, Suzuki M. Protamines. Isolation, characterization, structure and function. Mol Biol Biochem Biophys. 1973;12:1\\u0026ndash;114.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePugsley MK. \\u003cem\\u003eProtamine\\u003c/em\\u003e, in \\u003cem\\u003eMeyler's Side Effects of Drugs\\u003c/em\\u003e A. J.K., Editor. 2016. p. 1032\\u0026ndash;1034.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDomard A. pH and c.d. measurements on a fully deacetylated chitosan: application to CuII\\u0026mdash;polymer interactions. Int J Biol Macromol. 1987;9:98\\u0026ndash;104.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWillliam DW. Performance tests for the measurement of pH with glass electrodes in low ionic strength solutions including natural waters. Anal Chem. 1985;57(13):2567\\u0026ndash;70.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDeleebeeck L et al. \\u003cem\\u003eUnified pH Measurements of Ethanol, Methanol, and Acetonitrile, and Their Mixtures with Water\\u003c/em\\u003e. Sens (Basel), 2021. 21(11).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGarcia-Garcia P, et al. Nanoparticle-mediated selective Sfrp-1 silencing enhances bone density in osteoporotic mice. J Nanobiotechnol. 2022;20(1):462.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDao TPT, To NTH, Ho VV, Nguyen TH, Dang TA. A new formulation of curcumin using poly (lactic-co-glycolic acid)\\u0026mdash;polyethylene glycol diblock copolymer as carrier material. Adv Nat Sci: Nanosci Nanotechnol. 2014;5:035013.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJeon S et al. \\u003cem\\u003eSurface Charge-Dependent Cellular Uptake of Polystyrene Nanoparticles\\u003c/em\\u003e. Nanomaterials (Basel), 2018. 8(12).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBottaro E et al. \\u003cem\\u003eAnalysis of the Diffusion Process by pH Indicator in Microfluidic Chips for Liposome Production\\u003c/em\\u003e. Micromachines (Basel), 2017. 8(7).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGimondi S, et al. Microfluidic Devices: A Tool for Nanoparticle Synthesis and Performance Evaluation. ACS Nano. 2023;17(15):14205\\u0026ndash;28.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEdwards SA, Williams DR. Double layers and interparticle forces in colloid science and biology: analytic results for the effect of ionic dispersion forces. Phys Rev Lett. 2004;92(24):248303.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAbdelwahed W, et al. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev. 2006;58(15):1688\\u0026ndash;713.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFalahati M, et al. A health concern regarding the protein corona, aggregation and disaggregation. Biochim Biophys Acta Gen Subj. 2019;1863(5):971\\u0026ndash;91.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCaracciolo G, Farokhzad OC, Mahmoudi M. \\u003cem\\u003eBiological Identity of Nanoparticles In Vivo: Clinical Implications of the Protein Corona.\\u003c/em\\u003e Trends Biotechnol, 2017. 35(3): p. 257\\u0026ndash;264.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePederzoli F et al. \\u003cem\\u003eProtein corona and nanoparticles: how can we investigate on?\\u003c/em\\u003e. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2017. 9(6).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHu Y, et al. Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability. Acta Biomater. 2015;28:149\\u0026ndash;59.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSuk JS et al. \\u003cem\\u003ePEGylation as a strategy for improving nanoparticle-based drug and gene delivery.\\u003c/em\\u003e Adv Drug Deliv Rev, 2016. 99(Pt A): p. 28\\u0026ndash;51.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhu X, et al. Surface De-PEGylation Controls Nanoparticle-Mediated siRNA Delivery In Vitro and In Vivo. Theranostics. 2017;7(7):1990\\u0026ndash;2002.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCardarelli F, et al. Cholesterol-dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO living cells. Mol Pharm. 2012;9(2):334\\u0026ndash;40.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOttonelli I et al. \\u003cem\\u003eMicrofluidic Technology for the Production of Hybrid Nanomedicines\\u003c/em\\u003e. Pharmaceutics, 2021. 13(9).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSanthanes D, et al. Microfluidic formulation of lipid/polymer hybrid nanoparticles for plasmid DNA (pDNA) delivery. Int J Pharm. 2022;627:122223.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAnderluzzi GP. Y, \\u003cem\\u003eMicrofluidic Manufacture of Solid Lipid Nanoparticles: A Case Study on Tristearin-Based Systems\\u003c/em\\u003e. Drug Deliv Lett, 2020. 10.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTahir N, et al. Microfluidic fabrication and characterization of Sorafenib-loaded lipid-polymer hybrid nanoparticles for controlled drug delivery. Int J Pharm. 2020;581:119275.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi Y et al. \\u003cem\\u003eSynthesis of Polymer-Lipid Nanoparticles by Microfluidic Focusing for siRNA Delivery\\u003c/em\\u003e. Molecules, 2016. 21(10).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther. 1997;4(9):961\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYang Y, et al. Binding efficacy and kinetics of chitosan with DNA duplex: The effects of deacetylation degree and nucleotide sequences. Carbohydr Polym. 2017;169:451\\u0026ndash;7.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWu CL, Chen W, Lin HL, Jeng TL. Self-Assembled Structure of the Binary Complex of DNA with Cationic Lipid. Macromolecules. 2004;37:4974\\u0026ndash;80.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRouco H et al. \\u003cem\\u003eA Traffic Light System to Maximize Carbohydrate Cryoprotectants' Effectivity in Nanostructured Lipid Carriers' Lyophilization\\u003c/em\\u003e. Pharmaceutics, 2021. 13(9).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWong CY, Al-Salami H, Dass CR. Lyophilisation Improves Bioactivity and Stability of Insulin-Loaded Polymeric-Oligonucleotide Nanoparticles for Diabetes Treatment. Volume 21. AAPS PharmSciTech; 2020. p. 108. 3.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSalatin S, Maleki Dizaj S, Yari A, Khosroushahi. Effect of the surface modification, size, and shape on cellular uptake of nanoparticles. Cell Biol Int. 2015;39(8):881\\u0026ndash;90.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTreuel L, et al. Physicochemical characterization of nanoparticles and their behavior in the biological environment. Phys Chem Chem Phys. 2014;16(29):15053\\u0026ndash;67.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChoi J, et al. Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophages. J Appl Toxicol. 2009;29(1):52\\u0026ndash;60.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKelf TA, et al. Non-specific cellular uptake of surface-functionalized quantum dots. Nanotechnology. 2010;21(28):285105.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRejman J, et al. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J. 2004;377(Pt 1):159\\u0026ndash;69.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSadhukha T, Prabha S. Encapsulation in nanoparticles improves anti-cancer efficacy of carboplatin. AAPS PharmSciTech. 2014;15(4):1029\\u0026ndash;38.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePatel S, et al. Brief update on endocytosis of nanomedicines. Adv Drug Deliv Rev. 2019;144:90\\u0026ndash;111.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eOliveira CL, et al. Characterization of polymeric nanoparticles for intravenous delivery: Focus on stability. Colloids Surf B Biointerfaces. 2017;150:326\\u0026ndash;33.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLiang XH, et al. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat Biotechnol. 2016;34(8):875\\u0026ndash;80.\\u003c/span\\u003e\\u003c/li\\u003e\\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\":\"info@researchsquare.com\",\"identity\":\"drug-delivery-and-translational-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ddtr\",\"sideBox\":\"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/ddtr/default.aspx\",\"title\":\"Drug Delivery and Translational Research\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"lipid-polymeric hybrid nanoparticles, microfluidics, gene therapy, nanoparticles stability, endosomal escape\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3977241/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3977241/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. In this study, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG2000 were produced by single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs are successfully freeze-dried using 2.5 and 5% trehalose for long-term storage. The LPHNPs produced by MF and SSN increase 1.8–3.2 fold GapmeR cell uptake, respectively. They also endosomally escape in approximately 80%. The developed LPHNPs will be useful for targeting gene therapies.\",\"manuscriptTitle\":\"Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-02-26 13:18:43\",\"doi\":\"10.21203/rs.3.rs-3977241/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Major Revisions Needed\",\"date\":\"2024-03-28T15:05:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2024-02-21T20:16:15+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-02-21T19:21:35+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-02-21T04:47:36+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Drug Delivery and Translational Research\",\"date\":\"2024-02-20T18:39:53+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"drug-delivery-and-translational-research\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ddtr\",\"sideBox\":\"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/ddtr/default.aspx\",\"title\":\"Drug Delivery and Translational Research\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"6e467dcc-2eb4-4a8a-8c3e-f9e594282447\",\"owner\":[],\"postedDate\":\"February 26th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-06-14T00:34:10+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-3977241\",\"link\":\"https://doi.org/10.1007/s13346-024-01644-4\",\"journal\":{\"identity\":\"drug-delivery-and-translational-research\",\"isVorOnly\":false,\"title\":\"Drug Delivery and Translational Research\"},\"publishedOn\":\"2024-06-13 00:34:10\",\"publishedOnDateReadable\":\"June 13th, 2024\"},\"versionCreatedAt\":\"2024-02-26 13:18:43\",\"video\":\"\",\"vorDoi\":\"10.1007/s13346-024-01644-4\",\"vorDoiUrl\":\"https://doi.org/10.1007/s13346-024-01644-4\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3977241\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3977241\",\"identity\":\"rs-3977241\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}