Gene Delivery Performance Research Articles

Development and Evaluation of a Novel Biodegradable Poly(amidoamine) with Bis(guanidinium) and Benzene Ring Structures for Enhanced Gene Delivery

Cationic polymer carriers have shown promise in non-viral gene delivery but face challenges such as low transfection efficiency and high toxicity. To address these issues, we synthesized a novel biodegradable poly(amidoamine) (PAA) cationic polymer, 4-(4-biguanide benzoylamino)butylamine-N,N’-bis(acryloyl)cystamine (PDAB-CBA). This polymer incorporates bis(guanidinium) groups and a hydrophobic benzene ring to enhance membrane affinity and hydrophobic interactions, respectively, thereby improving gene delivery performance. The inclusion of disulfide bonds in the polymer’s backbone imparts reductive response properties, allowing for degradation into small molecular weight fragments within cellular environments and reducing cytotoxicity. Our studies demonstrated that PDAB-CBA formed stable, uniform nanoparticles with plasmid DNA (pDNA), achieving an encapsulation efficiency of approximately 85% at a 6:1 weight ratio. The polyplexes had the average particle size of around 100 nm and the zeta potential between 25-35 mV, ensuring effective cellular uptake. PDAB-CBA/pDNA polyplexes exhibited higher transfection efficiency, with relative light units (RLU) greater than 1 × 105 at weight ratios of 6:1 to 24:1, compared to PEI/pDNA. Furthermore, PDAB-CBA demonstrated low cytotoxicity, maintaining cell viability above 80% at weight ratios below 24:1, and effectively protected genes against DNaseI degradation and heparin replacement. The primary endocytosis pathway for these polyplexes was clathrin-mediated endocytosis. Overall, PDAB-CBA exhibited superior gene delivery capabilities and a favorable safety profile, providing a new strategy for the design of efficient and low-toxicity gene delivery vectors.

MCT4 knockdown by tumor microenvironment-responsive nanoparticles remodels the cytokine profile and eradicates aggressive breast cancer cells

Breast cancer is a wide-spread threat to the women’s health. The drawbacks of conventional treatments necessitate the development of alternative strategies, where gene therapy has regained hope in achieving an efficient eradication of aggressive tumors. Monocarboxylate transporter 4 (MCT4) plays pivotal roles in the growth and survival of various tumors, which offers a promising target for treatment. In the present study, pH-responsive lipid nanoparticles (LNPs) based on the ionizable lipid,1,2-dioleoyl-3-dimethylammonium propane (DODAP), were designed for the delivery of siRNA targeting MCT4 gene to the breast cancer cells. Following multiple steps of characterization and optimization, the anticancer activities of the LNPs were assessed against an aggressive breast cancer cell line, 4T1, in comparison with a normal cell line, LX-2. The selection of the helper phospholipid to be incorporated into the LNPs had a dramatic impact on their gene delivery performance. The optimized LNPs enabled a powerful MCT4 silencing by ∼90 % at low siRNA concentrations, with a subsequent ∼80 % cytotoxicity to 4T1 cells. Meanwhile, the LNPs demonstrated a 5-fold higher affinity to the breast cancer cells versus the normal cells, in which they had a minimum effect. Moreover, the MCT4 knockdown by the treatment remodeled the cytokine profile in 4T1 cells, as evidenced by 90 % and ∼64 % reduction in the levels of TNF-α and IL-6; respectively. The findings of this study are promising for potential clinical applications. Furthermore, the simple and scalable delivery vector developed herein can serve as a breast cancer-targeting platform for the delivery of other RNA therapeutics.

Development of a stable Sf9 insect cell line to produce VSV-G pseudotyped baculoviruses.

Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus to pseudotype budded baculoviruses. It was obtained by inserting the VSV-G gene under the control of the very strong and infection-inducible pXXL promoter and was subsequently diluted to establish oligoclonal lines, which were selected by the fusogenic properties of VSV-G and its expression levels in infected cells and purified budded virions. Next, to enhance the performance of the cell line, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses were pseudotyped and the expression of the transgene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased across all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.

Cinchona Alkaloid Polymers Demonstrate Highly Efficient Gene Delivery Dependent on Stereochemistry, Methoxy Substitution, and Length.

Nucleic acid delivery with cationic polymers is a promising alternative to expensive viral-based methods; however, it often suffers from a lower performance. Herein, we present a highly efficient delivery system based on cinchona alkaloid natural products copolymerized with 2-hydroxyethyl acrylate. Cinchona alkaloids are an attractive monomer class for gene delivery applications, given their ability to bind to DNA via both electrostatics and intercalation. To uncover the structure-activity profile of the system, four structurally similar cinchona alkaloids were incorporated into polymers: quinine, quinidine, cinchonine, and cinchonidine. These polymers differed in the chain length, the presence or absence of a pendant methoxy group, and stereochemistry, all of which were found to alter gene delivery performance and the ways in which the polymers overcome biological barriers to transfection. Longer polymers that contained the methoxy-bearing cinchona alkaloids (i.e., quinine and quinidine) were found to have the best performance. These polymers exhibited the tightest DNA binding, largest and most abundant DNA-polymer complexes, and best endosomal escape thanks to their increased buffering capacity and closest nuclear proximity of the payload. Overall, this work highlights the remarkable efficiency of polymer systems that incorporate cinchona alkaloid natural products while demonstrating the profound impact that small structural changes can have on overcoming biological hurdles associated with gene delivery.

Baicalin-modified polyethylenimine for miR-34a efficient and safe delivery.

The security and efficiency of gene delivery vectors are inseparable for the successful construction of a gene delivery vector. This work provides a practical method to construct a charge-regulated, hydrophobic-modified, and functionally modified polyethylenimine (PEI) with effective gene delivery and perfect transfection performance through a condensation reaction, named BA-PEI. The carrier was shown to possess a favorable compaction of miRNAs into positively charged nanoparticles with a hydrodynamic size of approximately 100nm. Additionally, BA-PEI possesses perfect degradability, which benefits the release of miR-34a from the complexes. In A549 cells, the expression level of the miR-34a gene was checked by Western blotting, which reflects the transfection efficiency of BA-PEI/miR-34a. When miR-34a is delivered to the cell, the perfect anti-tumor ability of the BA-PEI/miR-34a complex was systematically evaluated with the suppressor tumor gene miR-34a system in vitro and in vivo. BA-PEI-mediated miR-34a gene transfection is more secure and effective than the commercial transfection reagent, thus providing a novel approach for miR-34a-based gene therapy.

Highly Efficient Messenger RNA Transfection of Hard-to-Transfect Cells using Carbon Nanodots.

Although messenger RNA (mRNA)-based therapeutics opened up new avenues for treating various diseases, intracellular delivery of mRNA is still challenging, especially to hard-to-transfect cells. For successful mRNA therapy, the development of a delivery vehicle that can effectively transport mRNA into cells is essential. In this study, we synthesized carbon nanodots (CNDs) as an efficient mRNA delivery vehicle via a one-step microwave-assisted method. CNDs easily formed complexes with mRNA molecules by electrostatic interactions, and the gene delivery performance of CNDs was highly effective in hard-to-transfect cells. Considering their outstanding transfection ability, CNDs are expected to be further applied for mRNA-based cellular engineering.

A new nano hyperbranched β-pinene polymer: Controlled synthesis and nonviral gene delivery

Recently, an extensive research effort has been directed toward the improvement of nonviral transfection vectors, such as polymeric materials. The macromolecular structure of polymers has a substantial effect on their transfection efficacy. In this context, the modern advances in polymer production techniques, such as the deactivation-enhanced radical atom transfer polymerization (DE-ATRP), have been fundamental for the synthesis of controlled architecture nanomaterials. In this study, hyperbranched poly(β-pinene)-PDMAEMA-PEGDMA nanometric copolymers were synthesised at high conversion via DE-ATRP using different concentrations of β-pinene for gene delivery applications. The structural characterization and the biological performance of the materials were investigated. The copolymers’ molar mass (10,434–16,438 mol l-1), dispersity, and conversion (90–95%) varied significantly with β-pinene proportion on the polymerizations. The polymer-gene complexes generated (280–110 nm) presented excellent solution stability due to the β-pinene segment on the copolymers. Gene delivery and transfection were highly dependent on the copolymer composition. The copolymers containing the highest β-pinene proportions exhibited the best results with high transfection effectivity. In conclusion, the incorporation of β-pinene in DMAEMA-PEGMA copolymer formulations is a renewable option to improve the materials’ branching ratio, polyplex stability, and gene delivery performance without causing cytotoxic effects.

Fluorination Promotes the Cytosolic Delivery of Genes, Proteins, and Peptides.

The cytosolic delivery of biomolecules such as genes, proteins, and peptides is of great importance for biotherapy but usually limited by multiple barriers during the process. Cell membrane with high hydrophobic character is one of the representative biological barriers for cytosolic delivery. The introduction of hydrophobic ligands such as aliphatic lipids onto materials or biomolecules could improve their membrane permeability. However, these ligands are lipophilic and tend to interact with the phospholipids in the membrane as well as serum proteins, which may hinder efficient intracellular delivery. To solve this issue, our research group proposed the use of fluorous ligands with both hydrophobicity and lipophobicity as ideal alternatives to aliphatic lipids to promote cytosolic delivery.In our first attempt, fluorous ligands were conjugated onto cationic polymers to increase their gene delivery efficacy. The fluorination dramatically increased the gene delivery performance at low polymer doses. In addition, the strategy greatly improved the serum tolerance of cationic polymers, which is critical for efficient gene delivery in vivo. Besides serum tolerance, mechanism studies revealed that fluorination increases multiple steps such as cellular uptake and endosomal escape. Fluorination also allowed the assembly of low-molecular-weight polymers and achieved highly efficient gene delivery with minimal material toxicity. The method showed robust efficiency for polymers, including linear polymers, branched polymers, dendrimers, bola amphiphilies, and dendronized polymers.Besides gene delivery, fluorinated polymers were also used for intracellular protein delivery via a coassembly strategy. For this purpose, two lead fluoropolymers were screened from a library of amphiphilic materials. The fluoropolymers are greatly superior to their nonfluorinated analogues conjugated with aliphatic lipids. The fluorous lipids are beneficial for polymer assembly and protein encapsulation, reduced protein denaturation, facilitated endocytosis, and decreased polymer toxicity compared to nonfluorinated lipids. The materials exhibited potent efficacy in therapeutic protein and peptide delivery to achieve cancer therapy and were able to fabricate a personalized nanovaccine for cancer immunotherapy. Finally, the fluorous lipids were directly conjugated to peptides via a disulfide bond for cytosolic peptide delivery. Fluorous lipids drive the assembly of cargo peptides into uniform nanoparticles with much improved proteolytic stability and promote their delivery into various types of cells. The delivery efficacy of this strategy is greatly superior to traditional techniques such as cell-penetrating peptides both in vitro and in vivo. Overall, the fluorination techniques provide efficient and promising strategies for the cytosolic delivery of biomolecules.

Combinatorial Polycation Synthesis and Causal Machine Learning Reveal Divergent Polymer Design Rules for Effective pDNA and Ribonucleoprotein Delivery.

The development of polymers that can replace engineered viral vectors in clinical gene therapy has proven elusive despite the vast portfolios of multifunctional polymers generated by advances in polymer synthesis. Functional delivery of payloads such as plasmids (pDNA) and ribonucleoproteins (RNP) to various cellular populations and tissue types requires design precision. Herein, we systematically screen a combinatorially designed library of 43 well-defined polymers, ultimately identifying a lead polycationic vehicle (P38) for efficient pDNA delivery. Further, we demonstrate the versatility of P38 in codelivering spCas9 RNP and pDNA payloads to mediate homology-directed repair as well as in facilitating efficient pDNA delivery in ARPE-19 cells. P38 achieves nuclear import of pDNA and eludes lysosomal processing far more effectively than a structural analogue that does not deliver pDNA as efficiently. To reveal the physicochemical drivers of P38’s gene delivery performance, SHapley Additive exPlanations (SHAP) are computed for nine polyplex features, and a causal model is applied to evaluate the average treatment effect of the most important features selected by SHAP. Our machine learning interpretability and causal inference approach derives structure–function relationships underlying delivery efficiency, polyplex uptake, and cellular viability and probes the overlap in polymer design criteria between RNP and pDNA payloads. Together, combinatorial polymer synthesis, parallelized biological screening, and machine learning establish that pDNA delivery demands careful tuning of polycation protonation equilibria while RNP payloads are delivered most efficaciously by polymers that deprotonate cooperatively via hydrophobic interactions. These payload-specific design guidelines will inform further design of bespoke polymers for specific therapeutic contexts.

Integration of [12]aneN3 and Acenaphtho[1,2-b]quinoxaline as non-viral gene vectors with two-photon property for enhanced DNA/siRNA delivery and bioimaging

Two-photon fluorescent Acenaphtho[1,2-b]quinoxaline (ANQ) and the hydrophilic di-(triazole-[12]aneN3) moieties were combined through an alkyl chain (ANQ-A-M) or a β-hairpin motif with two aromatic γ-amino acid residues (ANQ-H-M) to explore their capabilities for in vitro and in vivo gene delivery and tracing. ANQ-A-M and ANQ-H-M showed the same maximum absorption at 420 nm, and their fluorescent intensities around 650 nm were varied in different solvents and became poor in the protic solvents. Gel electrophoresis assays indicated that both compounds completely retarded the migration of pDNA at 20 μM in the presence of DOPE. However, the DNA condensation with ANQ-H-M was not reversible, and the particle size of the corresponding complexes were larger indicated from the SEM and DLS measurements. In vitro transfections indicated ANQ-A-M/DOPE achieved Luciferase and GFP expressions were to be 7.9- and 5.7-fold of those by Lipo2000 in A549 cells respectively. However, ANQ-H-M showed very poor transfection efficiency in Luciferase expression. With the help of single/two-photon fluorescence imaging it clearly demonstrated that the successful transfection of ANQ-A-M was attributed to its cellular uptake, apparent lysosomal escape, and reversible release of DNA; and the poor transfection of ANQ-H-M was resulted from the aggregation of the DNA complexes which prevented them from the cellular uptake, and also the strong binding ability which is not easy to release DNA. ANQ-A-M/DOPE also exhibited robust gene silencing (83% knockdown of Luciferase) and GFP expression (2.47-fold higher) efficiency compared with Lipo2000 in A549 and zebrafish, respectively. The work demonstrated that the linkage structure between fluorescent and di(triazole-[12]aneN3) played the important role for their gene delivery performance, and that ANQ-A-M represents a vector with the strong transfection efficiency in vitro and in vivo as well as the efficient real time bioimaging properties, which is potential for the development in biomedical research.

Gene silencing-mediated immune checkpoint blockade for tumor therapy boosted by dendrimer-entrapped gold nanoparticles

Immune checkpoint blockade (ICB) has been regarded as one promising approach for tumor immunotherapy. Here, we report a functional nanoplatform based on generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer-entrapped gold nanoparticles (Au DENPs) as a nonviral vector to deliver programmed death-ligand 1 (PD-L1) small interfering RNA (siPD-L1) for subsequent PD-L1 gene silencing-mediated tumor immunotherapy. In this work, G5 dendrimers with amine termini were partially decorated with methoxy polyethylene glycol ( m PEG) on their periphery, entrapped Au NPs within their interiors, and were eventually labeled with fluorescamine. The generated functional Au DENPs possess desired dispersibility in water and colloidal stability, satisfactory cytocompatibility after complexation with siPD-L1, and efficient gene delivery performance. Strikingly, the functional Au DENPs enabled the delivery of siPD-L1 to cancer cells to efficiently knock down the PD-L1 protein expression, thus boosting the ICB-based immunotherapy of a xenografted melanoma mouse tumor model with a tumor inhibition efficiency much higher than the PD-L1 antibody. The immune responses were also well demonstrated by downregulation of PD-L1 protein on the tumor cell surface and abundant distribution of CD8+ and CD4+ T cells in the infiltrating tumor tissue and spleen organ. The developed functional dendrimer-based nanoplatform may be promising to boost ICB-based immunotherapy of other tumor types.

Protein liposomes-mediated targeted acetylcholinesterase gene delivery for effective liver cancer therapy

BackgroundEffective methods to deliver therapeutic genes to solid tumors and improve their bioavailability are the main challenges of current medical research on gene therapy. The development of efficient non-viral gene vector with tumor-targeting has very important application value in the field of cancer therapy. Proteolipid integrated with tumor-targeting potential of functional protein and excellent gene delivery performance has shown potential for targeted gene therapy.ResultsHerein, we prepared transferrin-modified liposomes (Tf-PL) for the targeted delivery of acetylcholinesterase (AChE) therapeutic gene to liver cancer. We found that the derived Tf-PL/AChE liposomes exhibited much higher transfection efficiency than the commercial product Lipo 2000 and shown premium targeting efficacy to liver cancer SMMC-7721 cells in vitro. In vivo, the Tf-PL/AChE could effectively target liver cancer, and significantly inhibit the growth of liver cancer xenografts grafted in nude mice by subcutaneous administration.ConclusionsThis study proposed a transferrin-modified proteolipid-mediated gene delivery strategy for targeted liver cancer treatment, which has a promising potential for precise personalized cancer therapy.

Construction of a Novel Reactive Oxygen Species-responsive Cationic Copolymer and Its Performance in Gene Delivery

Construction of a Novel Reactive Oxygen Species-responsive Cationic Copolymer and Its Performance in Gene Delivery

New Insights into Biocompatible Iron Oxide Nanoparticles: A Potential Booster of Gene Delivery to Stem Cells

Gene delivery to stem cells is a critical issue of stem cells-based therapies, still facing ongoing challenges regarding efficiency and safety. Recent advances in the controlled synthesis of biocompatible magnetic iron oxide nanoparticles (IONPs) have provided a powerful nanotool for assisting gene delivery to stem cells. However, this field is still at an early stage, with well-designed and scalable IONPs synthesis highly desired. Furthermore, the potential risks or bioeffects of IONPs on stem cells are not completely figured out. Therefore, in this review, the updated researches focused on the gene delivery to stem cells using various designed IONPs are highlighted. Additionally, the impacts of the physicochemical properties of IONPs, as well as the magnetofection systems on the gene delivery performance and biocompatibility are summarized. Finally, challenges attributed to the potential impacts of IONPs on the biologic behaviors of stem cells and the large-scale productions of uniform IONPs are emphasized. The principles and challenges summarized in this review provide a general guidance for the rational design of IONPs-assisted gene delivery to stem cells.

Membrane lipids destabilize short interfering ribonucleic acid (siRNA)/polyethylenimine nanoparticles.

Cell entry of polymeric nanoparticles (NPs) bearing polynucleotides is an important stage for successful gene delivery. In this work, we addressed the influence of cell membrane lipids on the integrity and configurational changes of NPs composed of short interfering ribonucleic acid (siRNA) and polyethylenimine. We focused on NPs derived from two different PEIs, unmodified low molecular weight PEI and linoleic acid (LA)-substituted PEI, and their interactions with two membrane lipids (zwitterionic 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)). Our experiments showed that POPS liposomes interacted strongly with both types of NPs, which caused partial dissociation of the NPs. POPC liposomes, however, did not induce any dissociation. Consistent with the experiments, steered molecular dynamics simulations showed a stronger interaction between the NPs and the POPS membrane than between the NPs and the POPC membrane. Lipid substitution on the PEIs enhanced the stability of the NPs during membrane crossing; lipid association between PEIs of the LA-bearing NPs as well as parallel orientation of the siRNAs provided protection against their dissociation (unlike NPs from native PEI). Our observations provide valuable insight into the integrity and structural changes of PEI/siRNA NPs during membrane crossing which will help in the design of more effective carriers for nucleic acid delivery.

Facile Synthesis of a Carnosine‐Pendent Cationic Polymer via Free Radical Polymerization and Application in Gene Delivery

Abstractl‐carnosine is a bipeptide with varieties of biomedical benefits, but rarely reported as a component in structurally defined polymers due to the unavailability of isolatable monomers. In this report, a simple method to synthesize a carnosine‐derived methacrylamide in three steps is established and a neutral polymer with a narrow molecular weight distribution (Mn 7.6 kDa, Ð 1.2) and high structural regularity is prepared via free radical polymerization, and a carnosine‐pendent cationic homopolymer is finally achieved after trifluoroacetic acid‐mediated deprotection without a concern of impurities such as metals. The antioxidative activity, cytotoxicity, DNA‐binding capability, and gene delivery performance of the cationic polymer are also studied.

Synthesis of Structurally Defined Cationic Polythiophenes for DNA Binding and Gene Delivery.

Water-soluble conjugated polymers (WCPs) have prospective applications in the field of bioimaging, disease diagnosis, and therapy. However, the use of WCPs with controllability and regioregularity for bioapplications have scarcely been reported. In this work, we synthesized polythiophenes containing ester side chains (P3ET) via Kumada catalyst-transfer polycondensation (KCTP) and confirmed a quasi-"living" chain-growth mechanism. In addition, we obtained cationic regioregular polythiophenes (cPTs) by aminolysis of P3ET with varied chain lengths, and studied DNA binding capability and gene delivery performance. Benefiting from photocontrolled generation of intracellular reactive oxygen species (ROS), the cationic polythiophenes successfully delivered DNA into tumor cells without additional polymer species.

Statistical versus block fluoropolymers in gene delivery.

Fluoropolymers have shown great promise in non-viral gene delivery. The current fluoropolymers developed for gene delivery are synthesized by grafting fluoroalkyls or fluoroaromatics onto cationic polymers. To expand the family of fluoropolymers for the transduction of nucleic acids, new strategies to synthesize fluoropolymers are required. In this study, we synthesized both statistical and block copolymers of poly(2-dimethylaminoethyl methacrylate) (pDMAEMA) and poly(heptafluorobutyl methacrylate) (pHFMA) via reversible addition-fragmentation chain transfer polymerization, and the transfection efficiencies of the fluorocopolymers were evaluated. The statistical fluorocopolymer exhibited dramatically higher performance in gene delivery than the block one, which is attributed to more efficient and sustained DNA uptake by the transfected cells. Moreover, the statistical copolymer of DMAEMA and HFMA showed a fluorine effect in gene delivery, and its efficiency was much superior to non-fluorinated polymers. The results revealed the structure and activity relationships of fluoropolymers consisting of DMAEMA and HFMA, and provided a new insight to guide the design of fluoropolymers for efficient gene delivery.

Microneedle: The future of pharmaceutical and cosmeceutical delivery systems

Quantum dots are semiconducting nanocrystals with diameters between 2-10 nanometers. Strong signal brightness, resistance to photo bleaching, size-tunable emission and large absorption coefficient across a wide spectral range make them powerful agent against conventional organic fluorescence dyes used in bioimaging applications. In addition to their optical imaging ability, quantum dots have multifunctional properties such as functionalization with targeting ligands for site specific delivery and loading with therapeutic drugs, peptides or oligonucleotides for drug and gene delivery applications thanks to their high surface to volume ratio. However, most of the synthesized QD’s emit in the visible region and they contain heavy metals such as Cd, Pb or Hg, which have intrinsic toxicity to living organisms. Ag2S QDs developed in recent years are of great interest with their excellent biocompatibility and strong emission intensity in near infrared region (NIR) of optical spectrum, offering higher photon penetration depth, lower absorption and scattering of light by cellular components and lower auto-fluorescence in the living tissue compared to visible region. Yet, the Ag2S compositions reported are still limited to anionic and PEGylated ones. We have focused on the development of first cationic Ag2S QDs for combined action of optical imaging and gene therapy. Here, we will discuss the synthesis of PEI coated Ag2S QDs, characterization and applications. PEI coating itself usually do not provide luminescent QDs. Yet, combination with small thiolated molecules provide means to tune the emission wavelength and intensity. We will discuss the influence of 2-mercaptopropionic acid and l-cysteine contribution in this formulation. Further we will discuss the effect of the coating composition on surface charge, size and biocompatibility. Optical imaging and gene delivery performance of these particles will be demonstrated as well.

Achieving high gene delivery performance with caveolae-mediated endocytosis pathway by (l)-arginine/(l)-histidine co-modified cationic gene carriers

Developing new amphiphilic polymers with natural product moieties has been regarded as a promising way to achieve biocompatibility and certain biological functions. In prior work, we developed some natural (l)-arginine modified cationic polymers (PAHMAA-Rs) as cationic gene carriers. For the sake of continuing optimize the gene delivery performance, herein, a new series of (l)-arginine and (l)-histidine co-modified cationic poly (ω-aminohexyl methacrylamide)s (PAHMAA-R-H) were synthesized and characterized with 1H NMR, GPC-SLS and FT-IR. Their proton buffering capacities were studied by acid-base titration assay. pDNA binding affinity and self-assembly properties of the polyplexes were analyzed by agarose gel retardation assay, DLS and AFM, respectively. In vitro cytotoxicity of the PAHMAA-R-H was determined by MTT and LDH assays in H1299 cells, the gene transfection efficacy and intracellular uptake capability were evaluated by luciferase assay and FACS, respectively. Moreover, the endocytosis pathways and intracellular distribution of the polyplexes were investigated by using specific endocytic inhibitors and fluorescent co-localization techniques. The results demonstrated that co-modification of (l)-arginine and (l)-histidine onto the PAHMAA polymer could enhance proton buffering capacity, shield surface charge, decrease cytotoxicity, and improve gene transfection efficiency and serum-compatibility. Moreover, the gene transfection and intracellular uptake behaviors were disclosed strongly rely on the (l)-arginine/(l)-histidine modification ratios. The polyplexes tend to be internalized through caveolae-mediated endocytosis gateway and localized with endosomes/lysosomes in H1299 cells. Notably, among the polymers, the PAHMAA-R18-H6 exhibited remarkable gene delivery efficiency and serum compatibility, which made it promising gene transfection agent for practical application.