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. 2025 Mar;15(3):908-924.
doi: 10.1007/s13346-024-01644-4. Epub 2024 Jun 13.

Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy

Daniel González-García  1   2 , Olga Tapia  2   3 , Carmen Évora  1   2 , Patricia García-García  4   5 , Araceli Delgado  6   7
Affiliations

Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy

Daniel González-García et al. Drug Deliv Transl Res. 2025 Mar.

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. The primary goal of this study is to develop formulations aimed to enhance the therapeutic potential of GapmeRs through technological approaches. To this end, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG2000 were produced by conventional 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 were successfully freeze-dried using trehalose in the range of 2.5-5% as cryoprotectant The LPHNPs produced by MF and SSN increase, 6 and 12 fold respectively, GapmeR cell uptake, and both of them reduce by 60-70% expression of Tob1 in 48 h.Our study demonstrates the efficacy of the developed LPHNPs as carriers for oligonucleotide delivery, offering valuable insights for their scale up production from a conventional bulk methodology to a high-throughput microfluidic technology.

Keywords: Endosomal escape; Gene therapy; Lipid-polymeric hybrid nanoparticles; Microfluidics; Nanoparticles stability.

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Conflict of interest statement

Declarations. Ethics approval: Not applicable. Competing interests: The authors have non-financial interests to disclose.

Figures

Fig. 1
Fig. 1
Enlarged perspective of the micromixer chip captured with stereomicroscope at 7.8 magnifications
Fig. 2
Fig. 2
Physicochemical characteristics of LPHNPs elaborated by conventional bulk single-step nanoprecipitation (SSN) using DOPE-mPEG2000 and DC-Chol (molar ratio 1:3) with protamine as a complexing agent. (A) Average size (bars) and ZP (symbol) and (B) Polydispersion index (PdI). Dashed line indicated values of 200 nm and PdI = 0.3 in the respective graph and dotted line 0 mV. n = 3
Fig. 3
Fig. 3
Physicochemical characteristics of LPHNPs elaborated by microfluidic (MF) using DOPE-mPEG2000 and DC-Chol (molar ratio 1:3) with protamine as a complexing agent. Flow Rate Ratio (FRR) of 3 and Total Flow Rate (TFR) of 0.8 mL/min. (A) Average size (bars) and ZP (symbol) and (B) Polydispersion index (PdI). Dashed line indicated values of 200 nm and PdI = 0.3 in the respective graph and dotted line 0 mV. n = 3
Fig. 4
Fig. 4
Images from LPHNPs made by (A) conventional single-step nanoprecipitation (SSN) and (B) microfluidics (MF)
Fig. 5
Fig. 5
GapmeR release profile from LPHNPs (A) at different pHs, 7.4 and 5.5 in 10% FBS (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
Fig. 6
Fig. 6
(A) Sizes and (B) PdI of LPHNPs in different stabilizers: 0.9% NaCl, 2.5% trehalose and 10% trehalose at time 0 and 24 h at 4 °C. 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 < 0.05
Fig. 7
Fig. 7
Increase 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 h 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 < 0.05
Fig. 8
Fig. 8
(A) Sizes and (B) 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 < 0.05
Fig. 9
Fig. 9
(A-L) Representative C2C12 cells exposed to naked GapmeR-FAM (A-D); (E-H) LPHNPs loaded with GapmeR-FAM elaborated by bulk SSN; (I-L) LPHNPs loaded with GapmeR-FAM elaborated by MF. Each representative confocal image is split into four sections: green channel representing FAM-GapmeR signal; red channel representing the late endosomal-lysosomal compartment labeled with the biomarker Lysotracker Red; “Merge” shows the green, red and blue (nuclei labeled with DAPI) signals; “Overlap” represents the colocalization overlays revealing as white dots those areas with GapmeR-FAM and Lysotracker Red positive signals. “Overlap” representations were obtained using the BIOP JACoP. (M) Quantification of the internalization ratio of GapmeR-FAM in cells exposed to LPHNPs prepared by bulk SSN or MF was calculated as the mean number of cytoplasmic FAM-positive foci per cell normalized to cells exposed to naked GapmeR-FAM (free). (N) Quantification using the BIOP JACoP plugin from FIJI of the percentage of GapmeR-FAM area that localizes within the endolysosomal Lysotracker-positive compartment (green-positive/red-positive “Overlap” area, colored bars) or that is distributed through the cytoplasm (green-positive/red-negative area, white bars) relativized to the total GapmeR-FAM area (total green-positive area, 100% bar). (O) Analysis of relative Tob1 mRNA fold levels 48 h after treatment of C2C12 cells with either control (white bars) or anti-Tob1 GapmeRs (colored bars) delivered by Dharmafect-1 transfection (DF) or HLPNPs elaborated by bulk SSN or MF as indicated. Scale bar: 10 μm (A-L); (*, **, ***) denotes statistically significant differences (p < 0.05, p < 0.005, p < 0.0005) with the naked GapmeR-FAM or among bulk SSN and MF, as indicated
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