Liposome Encapsulation of Oncolytic Virus M1 To Reduce Immunogenicity and Immune Clearance in Vivo.

Oncolytic viral therapy is an attractive novel strategy for cancer therapy. As a natural alphavirus, oncolytic virus M1 is able to infect and kill various zinc finger antiviral protein (ZAP)-deficient tumor cells selectively, while leaving normal cells undamaged. However, M1 can trigger the production of neutralizing antibodies that dramatically weaken its antitumor effect. In order to attenuate immunogenicity of the therapeutic M1 virus, we encapsulated it into liposomes (referred to as M-LPO) using the thin-film hydration method. The effect of anti-M1 neutralizing antibody on M-LPO was examined in LoVo and Hep 3B cell lines. In the absence of neutralizing antibodies, treating cells with naked M1, blank liposomes (LPO), M-LPO, or a simple mixture of M1 and liposomes (LPO+M1) inhibited cell growth. In the presence of neutralizing antibodies, only M-LPO inhibited cell growth. After intravenous administration, M-LPO reduced the production of the M1-neutralizing antibody and the corresponding immune response. Analysis of the M-LPO uptake by cells was examined by confocal microscopy using M1 labeled with FITC and liposomal shells labeled with RhB. The results suggest that M1 may be released from liposomes before or after M-LPO internalization. Taken together, our results suggest that encapsulating oncolytic virus M1 in liposomes may reduce intrinsic viral immunogenicity for improved anticancer therapy.

Highly stable polyglutamate derivatives/siRNA polyplex efficiently down-relegate survivin expression and augment the efficacy of cisplatin.

RNA interfere (RNAi)-based technology holds great promise in cancer treatment. The use of small interfering RNA (siRNA), however, is hampered by its low delivery efficiency in vivo when they are diluted in blood biofluids and in the presence of serum and salt. In this study, we developed the polyglutamate derivative polymer brush, poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (mPEG-b-PG-g-spermine, PPGS), which could efficiently deliver survivin-siRNA under ultra-high dilution and in the presence of salt (NaCl 150mM) and serum (10% FBS), most likely due to its PEG-shelled polymer brush structure. On the contrary, aggregation occurred when PEI/siRNA polyplex dispersed in saline and serum-containing media and PEI polyplex dissociated after making a 256-fold dilution. PPGS/si-survivin polyplex exhibited high cellular uptake efficiency and efficiently down-regulated the expression of survivin mRNA in the cisplatin-resistance of non-small cell human lung adenocarcinoma (A549/DDP) cells in the presence of serum. However, either PEI polyplex or Lipofectmine 2000 complex was unstable in serum and salt-containing media and at high dilution rates, which resulted in their dramatical decrease of cellular uptake and gene-silencing efficiency in these conditions. The PPGS/si-survivin polyplex also exhibited synergistic effects of killing the cancer cells by combination treatment with cisplatin. Therefore, the PPGS gene carrier showed great potential in systemic siRNA delivery, and its combination with chemotherapeutic drug is promising in treating drug resistant cancers.

Endothelial cell-targeted pVEGF165 polyplex plays a pivotal role in inhibiting intimal thickening after vascular injury.

Upregulation of vascular endothelial growth factor (VEGF) expression can inhibit intimal thickening after vascular injury. However, the lack of efficient gene delivery systems leads to insufficient VEGF expression, which prevents its application in gene therapy. In the present study, to improve the delivery of the plasmid vector with the VEGF gene (pVEGF165) to the injured vessel wall, we explored the potentially important difference between endothelial cell-targeted and nontargeted polymeric carriers. The αvß3 integrin is overexpressed on activated endothelial cells but not on normal quiescent vessels. In this study, CDG2-cRGD, synthesized by conjugating an αvß3 integrin-binding cyclic arginylglycylaspartic acid (cRGD) peptide with the Generation 2 polycation polyamidoamine (PAMAMG2)-g-cyclodextrin (termed as CDG2), was developed as a targetable carrier. It was observed that the specific integrin-ligand interactions greatly enhanced cellular internalization of CDG2-cRGD in human umbilical vein endothelial cells (HUVECs), which are notoriously difficult to transfect. Consequently, HUVECs were found to show remarkably high levels of VEGF165 expression induced by the CDG2-cRGD polyplex. Interestingly, VEGF165 overexpression in vivo was more complex than that in vitro, and in vivo assays demonstrated that the stimulus response to balloon injury in arteries could obviously upregulate VEGF165 expression in the saline-treated group, although it was not enough to prevent intimal thickening. In gene-transfected groups, intravascular delivery of pVEGF165 with the CDG2-cRGD polyplex into rabbits after vascular injury resulted in a significant inhibition of intimal thickening at 4 weeks, whereas the low therapeutic efficacy in the nontargeted CDG2-treated group was only comparable to that in the saline-treated group. It is becoming clear that the conflicting results of VEGF165 gene therapy in two gene-transfected groups are reflective of the pivotal role of the cRGD-conjugated carriers in achieving the beneficial therapeutic effects of vascular gene therapy.

Nanocarriers with tunable surface properties to unblock bottlenecks in systemic drug and gene delivery.

Nanocarrier-mediated drug and gene delivery systems hold great promise for providing more refined delivery (especially in cancer treatments) to maximize therapeutic efficacy while minimizing unfavorable side effects. Despite their promise, the highly effective transport of therapeutics in vivo remains a challenge. Over the last 20years, there has been a large amount of research directed toward the development of a multitude of nanocarriers for drug and gene delivery, but only a very small part has progressed into clinical trials. This suggests that the properties of current nanocarriers are not yet ideal for effective drug and gene delivery in vivo. Nanocarrier-mediated drug and gene delivery is a multi-step process, and inefficient delivery at any stage would ultimately result in an unsuccessful delivery. Unfortunately, existing nanocarriers with fixed surface properties, such as a PEGylated, cationized and bioconjugated surface, are not versatile enough to overcome the extracellular and intracellular barriers which require different surface properties. Consequently, their delivery efficacy is not optimal, leading to doubts and debates on the value of nanocarrier-based product development. To resolve the "fixed surface dilemma", the switchable surfaces of nanocarriers, which can surmount both extracellular and intracellular barriers, open up the possibility of highly efficient delivery in vivo. Here, we review and highlight the recent developments in the design of nanocarrier delivery systems with tunable surface properties in response to microenvironment triggers. Strategies including zwitterionic nanocarriers, polymer brushes, layer-by-layer nanocarriers and cleavable conjugated nanocarriers are presented. These representative examples and their respective outcomes elaborate the benefits and efficiencies of these nanocarriers at the individual stages of drug and gene delivery.

Salt-induced stability and serum-resistance of polyglutamate polyelectrolyte brushes/nuclear factor-κB p65 siRNA Polyplex enhance the apoptosis and efficacy of doxorubicin.

Short interfering RNAs (siRNAs) as chemotherapeutic RNAi agents hold great promise for a significant improvement in cancer therapy. Despite the promise, effective transport of siRNA with minimal side effects remains a challenge. The common problem associated with the low delivery efficiencies of current polycation-based gene delivery systems is their low stability in the presence of salt and serum. In the present study we developed the polyglutamate derivatives (PGS) polyelectrolyte brushes for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes/siRNA polyplex was colloidally stable (150 nm diameter) in physiological saline (150 mM NaCl), likely due to the osmotic brushes of PGS. The size-controlled siRNA/PGS polyplex also showed the serum resistance resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. The endothermic profile of ITC, their low values of Gibbs free energy and binding constants Kb under salt conditions provided the direct evidence that PGS polyelectrolyte brushes had a much lower binding affinity for serum proteins, compared with PEI 25KDa. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved efficient down-regulation of NF-κB p65 protein in HeLa cells. The NF-κB p65 down-regulation mediated by PGS polyelectrolyte brushes was more significant than PEI 25KDa and comparable to Lipofectamine 2000. Furthermore, the combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin demonstrated synergistic apoptotic and cytotoxic effects on HeLa cancer cells. The high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes/siRNA polyplex has potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies.