Abstract Gene delivery using cationic polymers has attracted much attention due to their potential advantages, such as large DNA loading capacity, ease of large-scale production, and reduced ...immunogenicity. We recently reported that polyplexes from polyN-N-(2-aminoethyl)-2-aminoethylaspartamide (PAsp(DET)), having an efficient endosomal escape due to pH-selective membrane destabilization, showed high transfection efficiency with minimal toxicity. Pharmacogenomic analysis demonstrated that PAsp(DET) also provided long-term security after transfection. We hypothesized that the biodegradability of PAsp(DET) played a significant role in achieving effective transfection. Gel permeation chromatography (GPC) and electrospray ionization mass spectrometry (ESI-MS) measurements of PAsp(DET) revealed their ability to undergo rapid degradation. In contrast, a derivative polycation, N-substituted polyglutamide (PGlu(DET)), showed no degradability, indicating that the degradation of PAsp(DET) was induced by a specific self-catalytic reaction between the PAsp backbone and the side-chain amide nitrogen. Degradation products of PAsp(DET) caused no cytotoxicity, even at high concentrations in the culture medium. Repeated transfection by administering the polyplexes for every 24 h showed that biodegradable PAsp(DET) provided a continuous increase in transgene expression, while non-degradable PGlu(DET) showed a decrease in transgene expression after 48 h, coupled with fluctuations in expression profiles of endogenous genes. In vivo intraperitoneal injection of PAsp(DET) induced minimal inflammatory cytokine induction to a level comparable to that of normal saline. These results indicate that the biodegradability of PAsp(DET) played a key role in achieving safe and sustained transgene expression, by minimizing cumulative toxicity caused by polycations remaining in cells or in the body.
PIC‐ing a winner: siRNA encapsulated by a phenylboronate‐functionalized polyion complex (PIC) micelle shows binding between the phenylboronate and the 3′ ribose of the siRNA (see scheme), stabilizing ...the complex under conditions equivalent to the extracellular environment. This complex is disrupted in response to addition of ATP, at a concentration comparable to that inside cells.
Protein‐free: A hydrogel containing phenylboronate was optimized so as to undergo rapid glucose‐dependent changes in the state of hydration under physiological aqueous conditions. A localized ...dehydration of the gel surface to form a “skin layer” enabled control of the release of insulin from the gel. This dehydration is induced by fluctuations in the glucose concentration in the range between normo‐ and hyperglycemia.
Gene therapy is a promising approach for treating diseases that are closely associated with excessive apoptosis, because the gene can effectively and sustainably introduce anti-apoptotic factors into ...cells. However, DNA delivery poses the risk of random genomic integration, leading to overexpression of the delivered gene and cancer development. Messenger RNA (mRNA) can evade integration events in target cells. We examined the use of mRNA-based therapeutics for introducing anti-apoptotic factors by using a mouse model of fulminant hepatitis. For introducing mRNA into the liver, a synthesised polymer-based carrier of polyplex nanomicelles was used for hydrodynamic intravenous injection. Using GFP as a reporter, we demonstrate that mRNA delivery induced efficient protein expression in almost 100% of liver cells, while plasmid DNA (pDNA) delivery provided a smaller percentage of GFP-positive cells. Analyses using Cy5-labelled mRNA and pDNA revealed that efficient expression by mRNA was attributed to a simple intracellular mechanism, without the need for nuclear entry. Consistent with this observation, Bcl-2 mRNA was more effective on reducing apoptosis in the liver of mice with fulminant hepatitis than Bcl-2 pDNA. Therefore, mRNA-based therapeutics combined with an effective delivery system such as polyplex nanomicelles is a promising treatment for intractable diseases associated with excessive apoptosis.
Ligand-mediated targeting of nanocarriers to tumors is an attractive strategy for increasing the efficiency of chemotherapies. Sialylated glycans represent a propitious target as they are broadly ...overexpressed in tumor cells. Because phenylboronic acid (PBA) can selectively recognize sialic acid (SA), herein, we developed PBA-installed micellar nanocarriers incorporating the parent complex of the anticancer drug oxaliplatin, for targeting sialylated epitopes overexpressed on cancer cells. Following PBA-installation, the micelles showed high affinity for SA, as confirmed by fluorescence spectroscopy even at intratumoral pH conditions, i.e., pH 6.5, improving their cellular recognition and uptake and enhancing their in vitro cytotoxicity against B16F10 murine melanoma cells. In vivo, PBA-installed micelles effectively reduced the growth rate of both orthotopic and lung metastasis models of melanoma, suggesting the potential of PBA-installed nanocarriers for enhanced tumor targeting
A series of the N-substituted polyaspartamides possessing repeating aminoethylene units in the side chain was prepared in this study to identify polyplexes with effective endosomal escape and low ...cytotoxicity. All cationic N-substituted polyaspartamides showed appreciably lower cytotoxicity than that of commercial transfection reagents. Interestingly, a distinctive odd–even effect of the repeating aminoethylene units in the polymer side chain on the efficiencies of endosomal escape and transfection to several cell lines was observed. The polyplexes from the polymers with an even number of repeating aminoethylene units (PA-Es) achieved an order of magnitude higher transfection efficiency, without marked cytotoxicity, than those of the polymers with an odd number of repeating aminoethylene units (PA-Os). This odd–even effect agreed well with the buffering capacity of these polymers as well as their capability to disrupt membrane integrity selectively at endosomal pH, leading to highly effective endosomal escape of the PA-E polyplexes. Furthermore, the formation of a polyvalent charged array with precise spacing between protonated amino groups in the polymer side chain was shown to be essential for effective disruption of the endosomal membrane, thus facilitating transport of the polyplex into the cytoplasm. These data provide useful knowledge for designing polycations to construct safe and efficient nonviral gene carriers.
Direct intracellular delivery of antibodies has gained much attention, although only a few agents have been developed, and none of them has reached clinical stages. The main obstacles here are the ...insufficient characteristics of delivery systems including stability and appropriate ability for intracellular antibody release. We tailored the structure of polyion complex (PIC) micelles by loading transiently charge-converted antibody derivatives for achieving enhanced stability, delivery to cytosol, and precise antigen recognition inside cells. Citraconic anhydride was used for the charge conversion of the antibody; the optimized degree of modification was identified to balance the stability of PIC micelles in the extracellular compartment and prompt pH-triggered disintegration after their translocation into the acidic endosomal compartment of target cells. The use of a mixture of homo- and block-catiomers in an appropriate ratio to construct PIC micelles substantially enhanced the endosomal escaping efficacy of the loaded antibody, leading to improved recognition of intracellular antigens.
Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the ...vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.
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A new type of polyion complex (PIC) micelle was prepared from lysozyme and the block copolymer, PEG-pAsp(EDA-Cit), that can switch the charge from anionic to cationic at the endosomal pH. The ...charge-conversion was due to the degradation of the citraconic amide side chain at pH 5.5. This abrupt charge-conversion can make the PIC micelles promptly release the internal protein in response to the endosomal pH. This pH-sensitive charge-conversion polymer is promising for the future design of nanocarriers for early endosomal release.
Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted ...polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd–even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.
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