Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab') fragments.

We reported previously the development of SMFv-PE38KDEL type I mutant (PE38KDEL-I; Mut-I), a recombinant immunotoxin in which a single-chain antibody derived from mouse SM5-1 monoclonal antibody is genetically fused to PE38KDEL-I. In comparison with the SMFv-PE38KDEL wild-type, Mut-I showed improved therapeutic efficacy and reduced toxicity. To overcome the problems associated with the immune response to the Pseudomonas exotoxin A (PE) component of Mut-I, we have constructed PE38KDEL-I-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with F(ab') fragments of a humanized SM5-1 monoclonal antibody (PE-NP-S). PE-NP-S specifically bound to SM5-1 binding protein-expressing hepatocellular carcinoma cell lines and was then internalized by these cells, resulting in significant cytotoxic effect. In SM5-1 binding protein-overexpressing tumor xenograft model, administration of PE-NP-S significantly inhibited tumor development and induced tumor regression. Moreover, PE-NP-S was shown to be much weaker in inducing vascular leakage syndrome in mice than Mut-I. The LD(50) of PE-NP-S was about 4-fold higher than that of Mut-I. Remarkably, PE-NP-S was of low immunogenicity in development of anti-PE neutralizing antibodies in vivo and was less susceptible to inactivation by anti-PE neutralizing antibodies compared with Mut-I. In conclusion, the resultant PE-NP-S possessed increased cancer therapeutic efficacy and had reduced nonspecific toxicity and immunogenicity, suggesting that it is a potential candidate in cancer therapy.

Preparation and Characterization of Paclitaxel-loaded PLGA nanoparticles coated with cationic SM5-1 single-chain antibody.

The purpose of this study was to develop paclitaxel-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles coated with cationic SM5-1 single-chain antibody (scFv) containing a polylysine (SMFv-polylys). SM5-1 scFv (SMFv) is derived from SM5-1 monoclonal antibody, which binds to a 230 kDa membrane protein specifically expressed on melanoma, hepatocellular carcinoma and breast cancer cells. SMFv-polylys was expressed in Escherichia coli and purified by cation-exchange chromatography. Purified SMFv-polylys was fixed to paclitaxel-loaded PLGA nanoparticles to form paclitaxel-loaded PLGA nanoparticles coated with SMFv-polylys (Ptx-NP-S). Ptx-NP-S was shown to retain the specific antigen-binding affinity of SMFv-polylys to SM5-1 binding protein-positive Ch-hep-3 cells. Finally, the cytotoxicity of Ptx-NP-S was evaluated by a non-radioactive cell proliferation assay. It was demonstrated that Ptx-NP-S had significantly enhanced in vitro cytotoxicity against Ch-hep-3 cells as compared with non-targeted paclitaxel-loaded PLGA nanoparticles. In conclusion, our results suggest that cationic SMFv-polylys has been successfully generated and may be used as targeted ligand for preparing cancer-targeted nanoparticles.

CD20 Antibody-Conjugated Immunoliposomes for Targeted Chemotherapy of Melanoma Cancer Initiating Cells.

Cancer initiating cells (CIC) are tumorigenic cancer cells that have properties similar to normal stem cells. CD20 is a phenotype of melanoma CIC that is responsible for melanoma drug resistance. Vincristine (VCR) is commonly used in melanoma therapy; however, it has been found ineffective against CIC. To target CD20+ melanoma CIC, we prepared VCR-containing immunoliposomes that were conjugated to CD20 antibodies (VCR-Lip-CD20). The drug release profile and the antibody-mediated targeting of the immunoliposomes were optimized to target CD20+ melanoma CIC. The immunoliposomes had desirable particle size (163 nm), drug encapsulation efficiency (91.8%), and drug release profile. We demonstrated that these immunoliposomes could successfully target more than 55% of CD20+ Chinese Hamster Ovary cells (CHO-CD20) even when the CHO-CD20 cells accounted for only 0.1% of a mixed population of CHO-CD20 and CHO cells. After treating WM266-4 melanoma mammospheres for 96 h, the ICo values of the drug delivered in VCR-Lip-CD20, VCR-Lip (VCR liposomes), and VCR were found to be 53.42, 98.99, and 99.09 µg/mL, respectively, suggesting that VCR-Lip-CD20 was 1.85 times more effective than VCR-Lip and VCR. VCR-Lip-CD20 could almost completely remove the tumorigenic ability of WM266-4 mammospheres in vivo, and showed the best therapeutic effect in WM266-4 melanoma xenograft mice. Significantly, VCR-Lip-CD20 could selectively kill CD20+ melanoma CIC in populations of WM266-4 cells both in vitro and in vivo. We demonstrated that VCR-Lip-CD20 has the potential to efficiently target and kill CD20+ melanoma CIC.

Polymer-lipid hybrid nanoparticles conjugated with anti-EGF receptor antibody for targeted drug delivery to hepatocellular carcinoma.

AIMS: The aim of this study was to obtain adriamycin-loaded polymer-lipid hybrid nanoparticles conjugated with anti-EGF receptor antibody (PLNP-Mal-EGFR) for hepatocellular carcinoma (HCC) chemotherapy. MATERIALS & METHODS: The nanoparticles were characterized by dynamic light scattering and fluorescence spectroscopy. The in vitro and in vivo distribution and anti-tumor activity of the nanoparticles were evaluated. RESULTS & CONCLUSION: PLNP-Mal-EGFR showed significantly enhanced cellular cytotoxicity against HCC cells overexpressing EGFR compared with nontargeted nanoparticles (polymer-lipid hybrid nanoparticles [containing DSPE-PEG-Mal] and polymer-lipid hybrid nanoparticles [containing DSPE-mPEG] combined with anti-EGFR Fab´). PLNP-Mal-EGFR and nontargeted nanoparticles could significantly reduce the proportion of side-population cells in HCC cells. The in vivo accumulation of PLNP-Mal-EGFR was obviously higher than that of nontargeted nanoparticles in SMMC-7721 HCC cells overexpressing EGFR. Notably, PLNP-Mal-EGFR showed significantly enhanced anti-tumor activity against HCC in vivo compared with nontargeted nanoparticles and free adriamycin. Therefore, PLNP-Mal-EGFR may serve as an effective therapeutic approach for HCC chemotherapy.

Inhibition of hepatocellular carcinoma growth using immunoliposomes for co-delivery of adriamycin and ribonucleotide reductase M2 siRNA.

The chemotherapy combined with gene therapy has received great attention. We developed targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab' co-delivering adriamycin (ADR) and ribonucleotide reductase M2 (RRM2) siRNA (ADR-RRM2-TLPD), to achieve combined therapeutic effects in human hepatocellular carcinoma (HCC) overexpressing EGFR. The antitumor activity and mechanisms of ADR-RRM2-TLPD were investigated. The results showed that RRM2 expression was higher in HCC than in non-HCC tissue, and RRM2 siRNA inhibited HCC cell proliferation, suggesting that RRM2 is a candidate target for HCC therapy. ADR-RRM2-TLPD delivered ADR and RRM2 siRNA to EGFR overexpressing HCC cells specifically and efficiently both in vitro and in vivo, resulting in enhanced therapeutic effects (cytotoxicity, apoptosis and senescence-inducing activity) compared with single-drug loaded or non-targeted controls, including ADR-NC-TLPD (targeted LPD co-delivering ADR and negative control siRNA), RRM2-TLPD (targeted LPD delivering RRM2 siRNA) and ADR-RRM2-NTLPD (non-targeted LPD co-delivering ADR and RRM2 siRNA). Mechanism studies showed that p21 is involved in the combined therapeutic effect of ADR-RRM2-TLPD. The average weight of the orthotopic HCC in mice treated with ADR-RRM2-TLPD was significantly lighter than that of mice treated with other controls. Thus, ADR-RRM2-TLPD represents a potential strategy for combined therapy of HCC overexpressing EGFR.

EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma.

The development of immunoliposomes for systemic siRNA (small interfering RNA) delivery is highly desired. We reported previously the development of targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab' (TLPD-FCC) for siRNA delivery, which showed superior gene silencing activity in EGFR-overexpressing breast cancers. However, TLPD-FCC did not achieve satisfactory gene silencing activity in EGFR-overexpressing hepatocellular carcinoma (HCC). In this study, some modifications including increased antibody conjugation efficiency and reduced PEGylation degree were made to TLPD-FCC to increase gene silencing activity in HCC. The resultant optimized liposomes denoted as TLPD-FP75 efficiently bound and delivered to EGFR-overexpressing HCC, resulting in enhanced gene silencing activity compared to untargeted LPD (NTLPD-FP75). Tissue distribution in vivo revealed that the accumulation of TLPD-FP75 was higher than NTLPD-FP75 in orthotopic HCC model of mice. The promoted uptake of TLPD-FP75 in HCC cells was confirmed by confocal microscopy. To investigate the in vivo gene silencing activity, we administered TLPD-FP75 by intravenous injections into mice bearing orthotopic HCC. The results showed TLPD-FP75 potently suppressed luciferase expression, while little silencing was observed in NTLPD-FP75. TLPD-FP75 was demonstrated to possess potent gene silencing activity in HCC and will potentially increase the feasibility of HCC gene therapy.

Novel dual-control poly(N-isopropylacrylamide-co-chlorophyllin) nanogels for improving drug release.

AIM: How to overcome insufficient drug release is an important issue in the drug-delivery system. MATERIALS & METHODS: Here, a novel temperature and UV dual-control poly(N-isopropylacrylamide [PNIPAM]-co-chlorophyllin) nanogel was prepared via the surfactant-free emulsion polymerization. RESULTS: The introduction of hydrophilic chlorophyllin to the PNIPAM chain backbone led to a narrow size of poly[NIPAM-co-CHLN nanogel (D ∼180 nm) confirmed by atomic force microscopy and transmission electron microscopy. This nanogel had a lower critical solution temperature (∼35°C), observed by dynamic laser light scattering. After the phase transition, the size under UV light (50 nm) was much smaller than that induced by temperature (90 nm). The inhomogeneous collapse was attributed to the temperature-gradient generated from the gel surface to the core with a surrounding dense PNIPAM layer. The obstacles that strongly inhibited 5-fluorouracil release was successfully overcome by light irradiation via a large drug diffusion coefficient. CONCLUSION: Consequently, the novel dual functional nanogel is potent for improving the drug-release profile.

Chemotherapy for gastric cancer by finely tailoring anti-Her2 anchored dual targeting immunomicelles.

Micelles with high in vivo serum stability and intratumor accumulation post intravenous (i.v.) injection are highly desired for promoting chemotherapy. Herein, we finely synthesized and tailored well-defined anti-Her2 antibody Fab fragment conjugated immunomicelles (FCIMs), which showed interesting dual targeting function. The thermosensitive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)(118) (PID(118)) shell with volume phase transition temperature (VPTT: 39 °C) and the anchored anti-Her2 Fab moiety contributed to the passive and active targeting, respectively. The doxorubicin (DOX) loading capacity of such FCIMs was successfully increased about 2 times by physically enhanced hydrophobicity of inner reservoir without structural deformation. The cellular uptake and intracellular accumulation of DOX by temperature regulated passive and antibody navigated active targeting was 4 times of Doxil. The cytotoxicity assay against Her2 overexpression gastric cancer cells (N87s) showed that the IC50 of the FCIMs was ≈ 9 times lower than that of Doxil under cooperatively targeting by Fab at T > VPTT. FCIMs showed high serum stability by increasing the corona PID(118) chain density (S(corona)/N(agg)). In vivo tissue distribution was evaluated in Balb/c nude mice bearing gastric cancer. As observed by the IVIS(®) imaging system, the intratumor accumulation of such finely tailored FCIMs system was obviously promoted 24 h post i.v. administration. Due to the high stability and super-targeting, the in vivo xenografted gastric tumor growth was significantly inhibited with relative tumor volume <2 which was much smaller than ≈ 5 of the control. Consequently, such finely tailored FCIMs with anti-Her2 active and temperature regulated passive dual tumor-targeting function show high potent in chemotherapy.

The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes.

The LPD (liposome-polycation-DNA complex) is an effective nanovector for systemically small interfering RNA (siRNA) delivery which was well characterized previously. However, little effort was spend on the development of targeted LPD conjugated with tumor specific antibody (TLPD) which would be potent in promoting siRNA delivery in tumor. Here, we prepared TLPD through a self-assembling process followed by anti-EGFR antibody conjugation. The effect of antibody type, conjugation strategy and amount on the physicochemical and biological properties of TLPD was investigated. We obtained optimized TLPD conjugated with anti-EGFR Fab' by conventional conjugation (TLPD-FCC), which possessed a small size around 150nm and superior in vitro stability. Compared with nontargeted LPD (NTLPD), TLPD-FCC showed significantly enhanced binding affinity and luciferase gene silencing activity in EGFR overexpressing MDA-MB-231 breast cancer cells in vitro. Moreover, the in vivo accumulation of TLPD-FCC was obviously higher than that of NTLPD in MDA-MB-231 tumor 24h post intravenous injection. The promoted uptake of TLPD-FCC in MDA-MB-231 tumor was further confirmed by confocal microscopy. Notably, three intravenous injections of siRNA in TLPD-FCC significantly silenced luciferase expression by ∼20%, whereas NTLPD showed little effect. All these results suggested that our TLPD-FCC have a great potential in delivering siRNA to EGFR overexpressing breast cancers.

Lyophilized HER2-specific PEGylated immunoliposomes for active siRNA gene silencing.

The development of a tumor-specific immunoliposome delivering small interfering RNA (siRNA) represents a practical way in cancer gene therapy. In this study, we developed PEGylated 3beta-[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol)/dioleoylphosphatidyl ethanolamine (DOPE) immunoliposomes conjugated with the Fab' of recombinant humanized anti-HER2 monoclonal antibody (PIL) for siRNA delivery. The results demonstrated that the lyophilized PIL (LPIL) prepared by the lyophilization/rehydration method possessed a significantly enhanced HER1 gene, a model target, silencing ability compared with PIL in HER2-overexpressing SK-BR3 cells. Among a series of LPIL with different PEGylation degree, LPIL containing 2.5%PEG (2.5%PEG LPIL) showed the best HER1 gene silencing activity. Confocal microscope studies demonstrated that 2.5%PEG LPIL could specifically bind to SK-BR3 cells and were sequentially internalized into them. Using RhoA as a cancer therapeutic target, 2.5%PEG LPIL entrapping anti-RhoA siRNA could specifically silence RhoA expression and inhibit cell invasion in SK-BR3 cells. In conclusion, these finding demonstrated the potential use of 2.5%PEG LPIL in specifically delivering siRNA to HER2-overexpressing cancers.