Nanoscale Polyplexes Research Articles

High Molecular Weight Chitosan-Complexed RNA Nanoadjuvant for Effective Cancer Immunotherapy.

Nucleic acid-based adjuvants have recently emerged as promising candidates for use in cancer vaccines to induce tumor-suppressing immune cells. In this study, we tested whether complexation of a nucleic acid-based adjuvant with chitosan (CTS) modulates immune adjuvant functions. As a nucleic acid-based adjuvant, we used toll-like receptor 3-recognizing RNA adjuvant (RA). Negatively charged RA formed nanoscale polyplexes with cationic CTS that possessed positive zeta potentials. RA/CTS polyplexes exerted dendritic cell (DC)-maturation effects without causing significant DC toxicity. This DC-maturation effect was CTS molecular weight dependent, with RA/CTS polyplexes with a CTS molecular weight of 340 kDa (RA/CTS 340K) producing the greatest effect. Subcutaneous injection of RA/CTS 340K polyplexes with the model tumor antigen ovalbumin exerted a preventive effect against challenge by ovalbumin-expressing tumor cells. It also provided greater inhibitory effects against a second challenge with the same tumor cells compared with other treatments. These protective effects of subcutaneous RA/CTS polyplex treatment were associated with the highest tumor antigen-specific humoral and cellular immune responses after tumor challenge, and with the greatest infiltration of CD4 helper T cell and CD8 T cell into the tumor tissues. Mice vaccinated with ovalbumin and RA/CTS polyplexes showed complete survival, even after repeated challenge with tumor cells. Our results suggest the potential of RA/CTS polyplexes as effective nanoadjuvants in the design of tumor vaccines and cancer immunotherapy.

Low molecular weight PEI-grafted carboxyl-modified soybean protein as gene carriers with reduced cytotoxicity and greatly improved transfection in vitro

SA-SP-PEI were synthesized via acylation reaction between carboxyl-modified soybean protein (SA-SP) and branched polyethylenimine (PEI) with molecular weight of 600, 1200, and 1800 Da, and designed as SA-SP-PEI600, SA-SP-PEI1200, and SA-SP-PEI1800, respectively. SA-SP-PEI could effectively condense plasmid DNA into nanoscale polyplexes and protect them from enzymatic digestion. MTT assay revealed that SA-SP-PEI exhibited reduced cytotoxicity on 293 T and SH-SY5Y cells. SA-SP-PEI1800/DNA complexes hold highest transfection efficiency on 293 T and SH-SY5Y cells with or without 10% serum, which was owing to its better serum stable and improved biocompatibility. Such polycationic soybean proteins have great potentials as gene carriers by further optimization.

Low molecular weight polyethylenimine-grafted soybean protein gene carriers with low cytotoxicity and greatly improved transfection invitro.

A series of gene carriers (SP-PEI) have been synthesized by acylation reaction between soybean protein and branched polyethylenimine with low molecular weight of 600, 1200 and 1800 Da, and designed as SP-PEI600, SP-PEI1200 and SP-PEI1800, respectively. SP-PEI could effectively condense plasmid DNA into nanoscale polyplexes with size range of 100-200 nm, and exhibited much lower cytotoxicity against 293T and SH-SY5Y cells than that of branched polyethylenimine (25 kDa). Invitro gene transfection demonstrated that SP-PEI/DNA complex displayed increased transfection against 293T and SH-SY5Y cells with the increase of the weight ratio of SP-PEI/DNA complex with or without 10% serum. At weight ratio of 24, SP-PEI1800/DNA polyplexes showed the highest transfection on SH-SY5Y cells, which was almost three folds higher than PEI (25 kDa). Furthermore, these SP-PEIs/DNA polyplexes could effectively transfect 293T and SH-SY5Y cells with or without 10% serum, suggesting their excellent serum tolerance.

Biologically responsive carrier-mediated anti-angiogenesis shRNA delivery for tumor treatment

Small interfering RNA (siRNA) has increased the hope for highly-efficient treatment of gene-related diseases. However, the stable and efficient delivery of therapeutic nucleic acids is a prerequisite for the successful clinical translation of RNA interfering therapy. To achieve this, we condensed the low molecular weight polyethyleneimine (PEI, Mw < 2000) with 2,6-pyridinedicarboxaldehyde (PDA) to synthesize a biologically responsive and degradable cationic polymer (abbreviated to PDAPEI) which was utilized as a gene vector for the delivery of a VEGF-A shRNA expression plasmid DNA (pDNA). The resulting electrostatic interaction between PDAPEI and pDNA led to the self-assembly of nanoscale polyplexes with suitable particle size and stable zeta potential. The PDAPEI/pDNA polyplexes demonstrated an outstanding gene transfection and silencing efficiency at 30 w/w ratio, as well as negligible cytotoxicity. Also, the designed polymer showed no stimulation to the innate immune system. Moreover, compared with PEI 25 KDa, the polyplexes accomplished comparatively better anti-angiogenesis efficacy, which resulted in the inhibition of tumor growth in subcutaneous tumor mice models. In conclusion, PDAPEI has great potential to be a gene delivery vector for cancer therapy.

APC targeted micelle for enhanced intradermal delivery of hepatitis B DNA vaccine

Chronic hepatitis B is a serious liver disease and puts people at high risk of death from cirrhosis and liver cancer. Although DNA vaccination has been emerged as a potential immunotherapeutic strategy for the treatment of chronic hepatitis B, the efficiencies were not adequate in clinical trials. Here we describe the design, synthesis, and evaluation of mannosylated phenylalanine grafted chitosan (Man-CS-Phe) as a DNA delivery vector for direct transfection of antigen presenting cells to improve cellular and humoral immunity to plasmid-coded antigen. The cationic Man-CS-Phe micelles condense plasmid DNA into nanoscale polyplexes and provide efficient protection of complexed DNA from nuclease degradation. The mannose receptor-mediated enhanced cell uptake and high in vitro transfection efficiency of the polyplexes were demonstrated in RAW 264.7 and DC 2.4 cells using GFP-expressing plasmid DNA. Furthermore, intradermal immunization of BALB/c mice indicated that hepatitis B DNA vaccine/Man-CS-Phe polyplexes not only induced multi-fold higher serum antibody titer in comparison to all other formulations including FuGENE HD, but also significantly stimulated T-cell proliferation and skewed T helper toward Th1 polarization. These results illustrate that the Man-CS-Phe can serve as a promising DNA delivery vector to harness both cellular and humoral arms of immune system.

New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes.

Release of siRNA from nanoscale polyplexes is a crucial yet little investigated process, important during all stages of therapeutic research. Here we develop new methods to characterize polyplex stability early on in the development of new materials. We used double fluorescent labeled siRNA to compare binding and stability of a panel of chemically highly diverse nanoscale polyplexes, including peptides, lipids, nanohydrogels, poly-L-lysine brushes, HPMA block copolymers and manganese oxide particles. Conventional EMSA and heparin competition methods were contrasted with a newly developed microscale thermophoresis (MST) assay, a near-equilibrium method that allows free choice of buffer conditions. Integrity of FRET-labeled siRNA was monitored in the presence of nucleases, in cell culture medium and inside living cells. This approach characterizes all relevant steps from polyplex stability, over uptake to in vitro knockdown capability. Diverging polyplex binding properties revealed drawbacks of conventional EMSA and heparin competition assays, where MST and FRET-based siRNA integrity measurements offered a better discrimination of differential binding strength. Since cell culture medium left siRNA in all polyplexes essentially intact, the relevant degradation events could be pinpointed to occur inside cells. Differential binding strength of the variegated polyplexes correlated only partially with intracellular degradation. The most successful compounds in RNAi showed intermediate binding strength in our assays. We introduce new methods for the efficient and informative characterization of siRNA polyplexes with special attention to stability. Comparing FRET-labeled siRNA in different polyplexes associates successful knockdown with intermediate siRNA stability in various steps from formulation to intracellular persistence.

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.

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.

A Molecular Dynamics Simulation Study on the Effect of Endogenous Molecules on SiRNA Polyplexes

Small interfering RNA (siRNA) based RNA interference (RNAi) is an important therapeutic strategy in treatment of cancer and hereditary diseases. RNAi is composed of several important stages, the first being siRNA delivery into cells. Polymeric carriers are used in delivery to aggregate siRNA molecules into nano-scale polyplexes. Upon delivery of polyplexes into cytosol, siRNA has to be released to trigger RNAi. However, mechanism of siRNA release from polyplexes is not clear; how endogenous molecules may affect the stability of polyplexes in cytosol is yet to be explored. In this study, we performed a series of molecular dynamics (MD) simulations to study the behavior of siRNA polyplexes in the presence of microRNA (miRNA), the most abundant nucleic acid inside cells. Polyethylenimine (PEI), known as a ‘gold-standard’ among polymeric delivery vehicles, was used as the carrier. miRNAs were introduced into pre-formed siRNA-PEI polyplexes. Quantitative analysis of the MD trajectories was carried out to characterize the change in the structure, compactness and charge distribution of the polyplex upon miRNA addition. PEI affinity toward siRNA and miRNA was determined and compared through individual simulations on siRNA-PEI and miRNA-PEI complexation. Data obtained from the MD simulations was compared with gel electrophoresis assays of siRNA-PEI-miRNA mixtures as well as siRNA-PEI-heparin mixtures; the latter served as a control due to the known capability of heparin to cause siRNA release. Our results revealed the ability of miRNAs to bind to pre-formed siRNA polyplexes through electrostatic interactions with PEI nitrogens. Therefore, upon the addition of miRNA, the size of the polyplex increased and its charge became negative. In agreement with the simulations, in gel electrophoresis, at the same concentrations, heparin was able to release siRNA while polyplexes maintained their integrity in the presence of miRNA.

Dextranation of Bioreducible Cationic Polyamide for Systemic Gene Delivery

A dextranated, bioreducible cationic polyamide was designed and employed for non-viral gene delivery in vitro and in vivo. Initially, a new bioreducible cationic polyamide with p-nitrophenyl ester terminal group (denoted as SSBAP) was synthesized by polycondensation reaction of an excess amount of bis-(p-nitrophenyl)-3, 3'-dithiodipropanoate and 1, 4-bis(3-aminopropyl)piperazine. The SSBAP was then chemically conjugated with 5 kDa amino-terminated dextran to yield dextran-SSBPA-dextran triblock copolymer (denoted as Dex-SSBAP-Dex). This copolymer was capable of binding genes to form nanoscale polyplexes with a near-neutral surface charge. Moreover, a sufficient gene release from the polyplexes in response to an intracellular reducing environment was observed. In vitro transfection against MCF-7 and SKOV-3 cells showed that Dex-SSBAP-Dex copolymer effectively transfected the cells with comparable efficiency to that of 2 5kDa branched or linear polyethylenimine as positive controls. Besides, intravenous administration of the copolymer-based polyplexes in nude mice afforded detectable gene expression largely in the lung. Importantly, the copolymer revealed low cytotoxicity in vitro, as determined by AlamarBlue assay, and caused no death of the mice. Dextranated, bioreducible cationic polyamide holds high potential as a non-viral vector for gene delivery.

Synthesis and characterization of new poly(ortho ester amidine) copolymers for non-viral gene delivery

A new type of pH-labile cationic polymers, poly(ortho ester amidine) (POEAmd) copolymers, has been synthesized and characterized with potential future application as gene delivery carriers. The acid-labile POEAmd copolymer was synthesized by polycondensation of a new ortho ester diamine monomer with dimethylaliphatimidates, and a non-acid-labile polyamidine (PAmd) copolymer was also synthesized for comparison using a triethylene glycol diamine monomer. Both copolymers were easily dissolved in water, and can efficiently bind and condense plasmid DNA at neutral pH, forming nano-scale polyplexes. The physicochemical properties of the polyplexes have been studied using dynamic light scattering, gel electrophoresis, ethidium bromide exclusion, and heparin competition. The average size of the polyplexes was dependent on the amidine:phosphate (N:P) ratio of the polymers to DNA. Polyplexes containing the acid-labile POEAmd or the non-acid-labile PAmd showed similar average particle size, comparable strength of condensing DNA, and resistance to electrostatic destabilization. They also share similar metabolic toxicity to cells as measured by MTT assay. Importantly, the acid-labile polyplexes undergo accelerated polymer degradation at mildly acid-pHs, resulting in increasing particle size and the release of intact DNA plasmid. Polyplexes from both types of polyamidines caused distinct changes in the scattering properties of Baby Hamster Kidney (BHK-21) cells, showing swelling and increasing intracellular granularity. These cellular responses are uniquely different from other cationic polymers such as polyethylenimine and point to stress-related mechanisms specific to the polyamidines. Gene transfection of BHK-21 cells was evaluated by flow cytometry. The positive yet modest transfection efficiency by the polyamidines (acid-labile and non-acid-labile alike) underscores the importance of balancing polymer degradation and DNA release with endosomal escape. Insights gained from studying such acid-labile polyamidine-based DNA carriers and their interaction with cells may contribute to improved design of practically useful gene delivery systems.

Subcutaneous Peripheral Injection of Cationized Gelatin/DNA Polyplexes As a Platform for Non-viral Gene Transfer to Sensory Neurons

Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.