Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages

A novel pH-sensitive and targetable antisense ODN delivery system based on multimolecular assembly into polyion complex (PIC) micelles of poly(L-lysine) (PLL) and a lactosylated poly(ethylene glycol)-antisense ODN conjugate (Lac-PEG-ODN) containing an acid-labile linkage (beta-propionate) between the PEG and ODN segments has been developed. The PIC micelles thus prepared had clustered lactose moieties on their peripheries and achieved a significant antisense effect against luciferase gene expression in HuH-7 cells (hepatoma cells), far more efficiently than that produced by the nonmicelle systems (ODN and Lac-PEG-ODN) alone, as well as by the lactose-free PIC micelle. In line with this pronounced antisense effect, the lactosylated PIC micelles showed better uptake than the lactose-free PIC micelles into HuH-7 cells; this suggested the involvement of an asialoglycoprotein (ASGP) receptor-mediated endocytosis process. Furthermore, a significant decrease in the antisense effect (27 % inhibition) was observed for a lactosylated PIC micelle without an acid-labile linkage (thiomaleimide linkage); this suggested the release of the active (free) antisense ODN molecules into the cellular interior in response to the pH decrease in the endosomal compartment is a key process in the antisense effect. Use of branched poly(ethylenimine) (B-PEI) instead of the PLL for PIC micellization led to a substantial decrease in the antisense effect, probably due to the buffer effect of the B-PEI in the endosome compartment, preventing the cleavage of the acid-labile linkage in the conjugate. The approach reported here is expected to be useful for the construction of smart intracellular delivery systems for antisense ODNs with therapeutic value.     

Therapeutic potential of a cell penetrating peptide (CPP,NP1) mediated siRNA delivery: Evidence in 3D spheroids of colon cancer cells

RNA interference holds great potential for cancer therapeutics and its success is highly dependent on an effective delivery system. As most preclinical drug screening in vitro was conducted in flat monolayer cell cultures, development of more physiologically relevant models is needed to enhance testing reliability and effectiveness. Here, the aim was to develop 3D cell spheroids and evaluate the efficiency of NP1, a novel cell penetrating peptide, CPP (STR-H16R8), developed by our group to assist siRNA delivery. NP1 elicited significant cellular uptake of siRNA and promoted great siRNA knockdown efficiency of Bcl-2 and VEGF mRNA in 3D spheroids (53% and 51%, respectively), induced marked apoptosis after silencing HIF mRNA, and 3D spheroids displayed apoptosis resistance compared to 2D cells. Taken together, 3D spheroids provide an improved model for testing siRNA delivery and NP1 has proved to be a powerful in vitro transfection reagent.     

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis,Characterization, and Evaluation

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; M_W 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.     

A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol)-block-poly(disulfide histamine) Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter) with almost neutral surface charge (+(5–10) mV). Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment), but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment). The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment). In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0). Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.     

Synthesis of PEGylated fullerene-5-fluorouracil conjugates to enhance the antitumor effect of 5-fluorouracil

Many drugs have been delivered by different types of nanoscale vehicles to enhance their therapeutic efficacy. 5-Fluorouracil (5FU) is a widely used antitumor drug, however its bioavailability still needs to be improved. Herein we synthesized a polyethylene glycol monomethylether-C(60)-5FU conjugate (mPEG-C(60)-5FU) and evaluated its antitumor efficacy in vitro. The results show that the inhibition abilities of mPEG-C(60)-5FU to the human breast cancer cell line MCF-7 and the human gastric carcinoma cell line BGC-823 are significantly higher than that of 5FU. The conjugate has good stability in murine serum for at least 24 h. Moreover, the PEGylated fullerene (mPEG-C(60)) vehicle is non-toxic to MCF-7 cells. These results demonstrate that mPEG-C(60) is an efficient vehicle for the delivery of 5FU.     

EGFR Transgene Stimulates Spontaneous Formation of MCF7 Breast Cancer Cells Spheroids with Partly Loss of HER3 Receptor

Multicellular spheroids with 3D cell–cell interactions are a useful model to simulate the growth conditions of cancer. There is evidence that in tumor spheroids, the expression of various essential molecules is changed compared to the adherent form of cell cultures. These changes include growth factor receptors and ABC transporters and result in the enhanced invasiveness of the cells and drug resistance. It is known that breast adenocarcinoma MCF7 cells can spontaneously form 3D spheroids and such spheroids are characterized by high expression of EGFR/HER2, while the natural phenotype of MCF7 cells is EGFR^low/HER2^low. Therefore, it was interesting to reveal if high epidermal growth factor receptor (EGFR) expression is sufficient for the conversion of adherent MCF7 to spheroids. In this study, an MCF7 cell line with high expression of EGFR was engineered using the retroviral transduction method. These MCF7-EGFR cells assembled in spheroids very quickly and grew predominantly as a 3D suspension culture with no special plates, scaffolds, growth supplements, or exogenous matrixes. These spheroids were characterized by a rounded shape with a well-defined external border and 100 µM median diameter. The sphere-forming ability of MCF7-EGFR cells was up to 5 times stronger than in MCF7^wt cells. Thus, high EGFR expression was the initiation factor of conversion of adherent MCF7^wt cells to spheroids. MCF7-EGFR spheroids were enriched by the cells with a cancer stem cell (CSC) phenotype CD24^−/low/CD44⁻ in comparison with parental MCF7^wt cells and MCF7-EGFR adhesive cells. We suppose that these properties of MCF7-EGFR spheroids originate from the typical features of parental MCF7 cells. We showed the decreasing of HER3 receptors in MCF7-EGFR spheroids compared to that in MCF^wt and in adherent MCF7-EGFR cells, and the same decrease was observed in the MCF7^wt spheroids growing under the growth factors stimulation. To summarize, the expression of EGFR transgene in MCF7 cells stimulates rapid spheroids formation; these spheroids are enriched by CSC-like CD24⁻/CD44⁻ cells, they partly lose HER3 receptors, and are characterized by a lower potency in drug resistance pomp activation compared to MCF7^wt. These MCF7-EGFR spheroids are a useful cancer model for the development of anticancer drugs, including EGFR-targeted therapeutics.     

Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges

RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design of novel exchangeable PEG-derivatized lipids. After systemic administration, these lipids can be released from the nanoparticle surface. Moreover, the design and synthesis of novel cationic lipids that are more fusogenic and the use of internalizing targeting ligands have contributed to the emergence of novel lipid-based nanoparticles with remarkable transfection efficiency.     

PEGylated Poly(3-hydroxybutyrate) Scaffold for Hydration-Driven Cell Infiltration,Neo-Tissue Ingrowth,and Osteogenic Potential

When poly(3-hydroxybutyrate) (PHB) scaffold was surface PEGylated with poly(ethylene glycol) methacrylate-block-polystyrene (PS-b-PEGMA), human MG63 osteoblast and HT1080 fibroblast unattached to the PEGylated PHB fibers, but diffusely infiltrated into the hydrated microfibrous scaffold matrix. These cells could grow at reduced cytoskeleton and proliferate within the spatially microenvironment, in which early buildup of neo-tissue construct was stimulated. Biofouling of PEGylated PHB scaffold was controlled, with a remarkably reduction of 90–98% in protein and biological cells adhesions. Besides retaining the original good cytocompatibility, this circumstance was able to activate osteoblast aggregates in the scaffold to up-regulate expression of osteogenesis-related genes, boosting osteogenic differentiation.     

Antitumor Effect of Regorafenib on MicroRNA Expression in Hepatocellular Carcinoma Cell Lines

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is one of the leading causes of cancer-related deaths worldwide. Regorafenib, a multi-kinase inhibitor, is used as a second-line treatment for advanced HCC. Here, we aimed to investigate the mechanism of the antitumor effect of regorafenib on HCC and evaluate altered microRNA (miRNA) expression. Cell proliferation was examined in six HCC cell lines (HuH-7, HepG2, HLF, PLC/PRF/5, Hep3B, and Li-7) using the Cell Counting Kit-8 assay. Xenografted mouse models were used to assess the effects of regorafenib in vivo. Cell cycle analysis, western blotting analysis, and miRNA expression analysis were performed to identify the antitumor inhibitory potential of regorafenib on HCC cells. Regorafenib suppressed proliferation in HuH-7 cell and induced G0/G1 cell cycle arrest and cyclin D1 downregulation in regorafenib-sensitive cells. During miRNA analysis, miRNA molecules associated with the antitumor effect of regorafenib were found. Regorafenib suppresses cell proliferation and tumor growth in HCC by decreasing cyclin D1 via alterations in intracellular and exosomal miRNAs in HCC.     

pH-triggered poly(ethylene glycol) nanogels prepared through orthoester linkages as potential drug carriers

The new acid-labile poly(ethylene glycol) (PEG) nanogels were prepared by copolymerization of a new crosslinking agent containing orthoester groups (OEAM) and methoxypolyethylene glycol acrylate (MPEGAC). DOX was loaded into PEG nanogels with a loading content of 18.2%, which was highly desirable for targeted cancer therapy without premature drug release in neutral environment. The cellular uptake and cytotoxicity of DOX-loaded PEG nanogels were measured using SH-SY5Y and HepG2 cells. Tumor penetration and antitumor activity were investigated using SH-SY5Y tumor-like spheroids. All results demonstrate that the pH-sensitive PEG nanogels may be used as potential drug carriers for chemotherapy.     

Bioreducible poly(amido amine) copolymers derived from histamine and agmatine for highly efficient gene delivery

Histamine (HIS) can facilitate the endosomal escape of polyplexes via the ‘proton sponge effect’ because of its imidazole groups. Agmatine (AGM) can improve the transmembrane process of polyplexes as a result of its guanidinium groups. Therefore, HIS and AGM were used as amino monomers to react with cystamine bisacrylamide (CBA) through Michael addition. The synthesized peptide‐mimicking poly(CBA‐HIS/AGM)s showed high transfection efficiency and low cytotoxicity, indicating their great potential as gene carriers. The results also demonstrated that the effects of HIS and AGM on the properties of poly(CBA‐HIS/AGM)s were different: HIS could increase their buffering capacities and bioreducibility, but AGM could facilitate their plasmid DNA packaging and condensing abilities. In addition, the results of transfection mechanism studies indicated that poly(CBA‐HIS/AGM) polyplexes entered into cells mainly via clathrin‐dependent endocytosis and they could efficiently escape the endosome, indicating endosomal escape was not the limiting step for gene delivery based on these polymers. © 2018 Society of Chemical Industry     

PEGylated block copolymers containing tertiary amine side-chains cleavable via acid-labile ortho ester linkages for pH-triggered release of DNA

A new type of acid-labile cationic copolymer consisting of a hydrophilic poly(ethylene glycol) (PEG) block and a polymethacrylamide block bearing tertiary amines linked by acid-labile ortho ester rings in side chains (PAOE), with defined chain length, had been synthesized via RAFT polymerization. The copolymers could efficiently bind and condense plasmid DNA at neutral pH into narrowly dispersed nano-scale polyplexes. The hydrolysis of ortho ester group in the side-chains of PAOE followed a distinct exocyclic mechanism and the rate of hydrolysis was much accelerated at mildly acidic pH, resulting in the accelerated disruption of polyplexes and the release of intact plasmid DNA. The three polymers were not toxic to cultured COS-7 cells as measured by MTT assay. As expected, PEG segments of the PEG-b-PAOE copolymers prevented nonspecific transfection of COS-7 cells. Once conjugated to a targeting ligand to enhance cell-specific entry, PEG-b-PAOE with its pH-triggered DNA release properties may achieve efficient intracellular delivery of DNA or other nucleic acid therapeutics.     

Effect of Relative Arrangement of Cationic and Lipophilic Moieties on Hemolytic and Antibacterial Activities of PEGylated Polyacrylates

Synthetic amphiphilic polymers have been established as potentially efficient agents to combat widespread deadly infections involving antibiotic resistant superbugs. Incorporation of poly(ethylene glycol) (PEG) side chains into amphiphilic copolymers can reduce their hemolytic activity while maintaining high antibacterial activity. Our study found that the incorporation of PEG has substantially different effects on the hemolytic and antibacterial activities of copolymers depending on structural variations in the positions of cationic centers relative to hydrophobic groups. The PEG side chains dramatically reduced the hemolytic activities in copolymers with hydrophobic hexyl and cationic groups on the same repeating unit. However, in case of terpolymers with cationic and lipophilic groups placed on separate repeating units, the presence of PEG has significantly lower effect on hemolytic activities of these copolymers. PEGylated terpolymers displayed substantially lower activity against Staphylococcus aureus (S. aureus) than Escherichia coli (E. coli) suggesting the deterring effect of S. aureus’ peptidoglycan cell wall against the penetration of PEGylated polymers. Time-kill studies confirmed the bactericidal activity of these copolymers and a 5 log reduction in E. coli colony forming units was observed within 2 h of polymer treatment.     

Hyperbranched polymeric "star vectors" for effective DNA or siRNA delivery

Although gene therapy offers an attractive strategy for treating inherited disorders, current techniques using viral and nonviral delivery systems have not yielded many successful results in clinical trials. Viral vectors such as retroviruses, lentiviruses, and adenoviruses deliver genes efficiently; however, the possibility of negative outcomes from viral transformation cannot be completely ruled out. In contrast, various types of nonviral vectors are attracting considerable attention because they are easier to handle and induce weak immune responses. Cationic polymers, such as polyethylenimine (PEI) and poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm), can generate nanoparticles through the formation of polyion complexes, "polyplexes" with DNA. These nonviral systems offer many advantages over viral systems. The primary obstacle to implementing these cationic polymers in an effective gene therapy remains their comparatively inefficient gene transfection in vivo. We describe four strategies for the development of hyperbranched star vectors (SVs) for enhancing DNA or siRNA delivery. The molecular design was performed by living radical polymerization in which the chain length can be controlled by photoirradiation and solution conditions, including concentrations of the monomer or iniferter (a molecule that serves as a combination of initiator, transfer agent, and terminator). The branch composition is controlled by the types of monomers that are added stepwise. In our first strategy, we prepared a series of only cationic PDMAPAAm-based SVs with no branches or 3, 4, or 6 branching numbers. These SVs could form polyion complexes (polyplexes) by mixing with DNA only in aqueous solution. The relative gene expression activity of the delivered DNA increased according to the degree of branching. In addition, increasing the molecular weight of SVs and narrowing their polydispersity index (PDI) improved their activity. For targeting DNA delivery to the specific cells, we modified the SV with ligands. Interestingly, the SV could adsorb the RGD peptide, making gene transfer possible in endothelial cells which are usually refractory to such treatments. The peptide was added to the polyplex solution without covalent derivatization to the SV. The introduction of additional branching by cross-linking using iniferter-induced coupling reactions further improved gene transfection activity. After block copolymerization of PDMAPAAm-based SVs with a nonionic monomer (DMAAm), the blocked SVs (BSVs) produced polyplexes with DNA that had excellent colloidal stability for 1 month, leading to efficient in vitro and in vivo gene delivery. Moreover, BSVs served as carriers for siRNA delivery. BSVs enhanced siRNA-mediated gene silencing in mouse liver and lung. As an alternative approach, we developed a novel gene transfection method in which the polyplexes were kept in contact with their deposition surface by thermoresponsive blocking of the SV. This strategy was more effective than reverse transfection and the conventional transfection methods in solution.     

Hemocompatible interface control via thermal-activated bio-inspired surface PEGylation

A facile thermal-activated self-copolymerization coating of antifouling layers was designed. PEGylated poly(ethyleneimine) (PEI-PEGMA) was first synthesized, mixed with an alkaline dopamine solution, and then concurrently codeposited onto versatile substrates of both organic and inorganic materials. Thermal-activated codeposition of dopamine/PEI-PEGMA could then proceed, giving rise to good hemocompatible performance in respect to low fibrinogen adsorption, ultra-low blood cell adhesion and activation, and almost undetectable hemolytic activities. This work also revealed that the hemocompatible performance was contributed by PEGylated PEI but not dopamine, as the poly(dopamine)-coating was shown to induce a high level of fibrinogen adsorption as well as vigorous platelet activation.     

Antibody Conjugated PLGA Nanocarriers and Superparmagnetic Nanoparticles for Targeted Delivery of Oxaliplatin to Cells from Colorectal Carcinoma

Anti-CD133 monoclonal antibody (Ab)-conjugated poly(lactide-co-glycolide) (PLGA) nanocarriers, for the targeted delivery of oxaliplatin (OXA) and superparamagnetic nanoparticles (IO-OA) to colorectal cancer cells (CaCo-2), were designed, synthesized, characterized, and evaluated in this study. The co-encapsulation of OXA and IO-OA was achieved in two types of polymeric carriers, namely, PLGA and poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) by double emulsion. PLGA_IO-OA_OXA and PEGylated PLGA_IO-OA_OXA nanoparticles displayed a comparable mean diameter of 207 ± 70 nm and 185 ± 119 nm, respectively. The concentration of the released OXA from the PEGylated PLGA_IO-OA_OXA increased very rapidly, reaching ~100% release after only 2 h, while the PLGA_IO-OA_OXA displayed a slower and sustained drug release. Therefore, for a controlled OXA release, non-PEGylated PLGA nanoparticles were more convenient. Interestingly, preservation of the superparamagnetic behavior of the IO-OA, without magnetic hysteresis all along the dissolution process, was observed. The non-PEGylated nanoparticles (PLGA_OXA, PLGA_IO-OA_OXA) were selected for the anti-CD133 Ab conjugation. The affinity of Ab-coated nanoparticles for CD133-positive cells was examined using fluorescence microscopy in CaCo-2 cells, which was followed by a viability assay.     

pH-responsive PEGylated nanogel containing platinum nanoparticles: Application to on–off regulation of catalytic activity for reactive oxygen species

Synthesis of pH-responsive PEGylated nanogels platinum nanoparticles (PtNPs: <2 nm) was successfully carried out through the reduction of K₂PtCl₆ within the PEGylated nanogels constructed from cross-linked poly[2-(N,N-diethylamino)ethyl methacrylate] (PDEAMA) core and tethered PEG chains. The resulting PEGylated nanogels containing PtNPs showed significant catalytic activity for reactive oxygen species (ROS) in response to skin-environmental pH (acid), whereas the almost no catalytic activity for ROS was observed at physiological pH due to the volume phase transition of the PDEAMA gel core. Thus, pH-responsive and PEGylated nanogel containing PtNPs can be utilized to the skin-specific ROS-scavengers for the skin aging.     

Low-Frequency Ultrasound Effects on Intracellular Barriers in Nonviral Gene Delivery Processes

Gene therapy, the expression in cells of genetic material with therapeutic activity, has emerged as a promising approach for the treatment or prevention of human diseases. At the present time, major somatic gene-transfer approaches employ either viral or nonviral vectors. Nonviral vectors are less efficient at introducing and maintaining foreign gene expression, but have the profound advantage of being nonpathogenic and nonimmunogenic. In this study, we aimed to develop an efficient nonviral gene delivery system in which low-frequency ultrasound (LFUS) was applied to enhance gene expression of polyplexes formed with poly(2-dimethylaminoethyl methacrylate) and plasmid encoding for green fluorescent protein. Ultrasound (US), and in particular LFUS, can cause temporary membrane permeabilization and thereby enhance drug and gene entrance into viable cells. We evaluated possible additional favorable effects of LFUS on the polyplex transfection process, such as overcoming intracellular barriers. We found that pDMAEMA protected the plasmid DNA from ultrasonic degradation. Atomic force microscopy analysis also confirmed that the LFUS did not change the polyplexes’ morphology. We also attained an insight into the structure of polyplexes during LFUS exposure and found that LFUS induced a temporary partial detachment between the polymer chains and the plasmid. In addition, LFUS application on ovarian carcinoma cells transfected with the polyplexes induced a 27% enhancement in transfection efficiency. Based on these results, we propose that LFUS enhances the decomplexation of the polyplexes, and therefore, can be used to optimize transfection efficiency.     

Random Copolymers of Lysine and Isoleucine for Efficient mRNA Delivery

Messenger RNA (mRNA) is currently of great interest as a new category of therapeutic agent, which could be used for prevention or treatment of various diseases. For this mRNA requires effective delivery systems that will protect it from degradation, as well as allow cellular uptake and mRNA release. Random poly(lysine-co-isoleucine) polypeptides were synthesized and investigated as possible carriers for mRNA delivery. The polypeptides obtained under lysine:isoleucine monomer ratio equal to 80/20 were shown to give polyplexes with smaller size, positive ζ-potential and more than 90% encapsulation efficacy. The phase inversion method was proposed as best way for encapsulation of mRNA into polyplexes, which are based on obtained amphiphilic copolymers. These copolymers showed efficacy in protection of bound mRNA towards ribonuclease and lower toxicity as compared to lysine homopolymer. The poly(lysine-co-isoleucine) polypeptides showed greater than poly(ethyleneimine) efficacy as vectors for transfection of cells with green fluorescent protein and firefly luciferase encoding mRNAs. This allows us to consider obtained copolymers as promising candidates for mRNA delivery applications.