Shuang Dou

Doxorubicin-Tethered Responsive Gold Nanoparticles Facilitate Intracellular Drug Delivery for Overcoming Multidrug Resistance in Cancer Cells

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. Through the development of a drug delivery system that tethers doxorubicin onto the surface of gold nanoparticles with a poly(ethylene glycol) spacer via an acid-labile linkage (DOX-Hyd@AuNPs), we have demonstrated that multidrug resistance in cancer cells can be significantly overcome by a combination of highly efficient cellular entry and a responsive intracellular release of doxorubicin from the gold nanoparticles in acidic organelles. DOX-Hyd@AuNPs achieved enhanced drug accumulation and retention in multidrug resistant MCF-7/ADR cancer cells when it was compared with free doxorubicin. It released doxorubicin in response to the pH of acidic organelles following endocytosis, opposite to the noneffective drug release from doxorubicin-tethered gold nanoparticles via the carbamate linkage (DOX-Cbm@AuNPs), which was shown by the recovered fluorescence of doxorubicin from quenching due to the nanosurface energy transfer between the doxorubicinyl groups and the gold nanoparticles. DOX-Hyd@AuNPs therefore significantly enhanced the cytotoxicity of doxorubicin and induced elevated apoptosis of MCF-7/ADR cancer cells. With a combined therapeutic potential and ability to probe drug release, DOX-Hyd@AuNPs represent a model with dual roles in overcoming MDR in cancer cells and probing the intracellular release of drug from its delivery system.

Sheddable Ternary Nanoparticles for Tumor Acidity-Targeted siRNA Delivery

Drug delivery systems for cancer therapy usually need to be sterically stabilized by a poly(ethylene glycol) (PEG) layer during blood circulation to minimize nonspecific interactions with serum components. However, PEGylation significantly reduces cellular uptake of the delivery systems after they accumulate at the tumor site, which markedly impairs the in vivo antitumor efficiency. Here, we develop a ternary small interfering RNA (siRNA) delivery system with tumor acidity-activated sheddable PEG layer to overcome the challenge. The sheddable nanoparticle is fabricated by introducing a tumor acidity-responsive PEGylated anionic polymer to the surface of positively charged polycation/siRNA complexes via electrostatic interaction. We show clear evidence that introducing the PEGylated anionic polymer to the surface of a nanoparticle markedly reduces its nonspecific interactions with protein. We further demonstrate that the nanoparticle is capable of deshielding the PEG layer at the slightly acidic tumor extracellular microenvironment to facilitate the delivery of siRNA to the tumor cells after accumulation at the tumor site. Accordingly, this promotes the RNA-interfering efficiencies and enhances the inhibition of tumor growth. Such delivery system with the ability to deshield the PEG layer at the target tissues has remarkable potential in cancer therapy.

Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy.

Delivery of small interfering RNA (siRNA) has been one of the major hurdles for the application of RNA interference in therapeutics. Here, we describe a cationic lipid assisted polymeric nanoparticle system with stealthy property for efficient siRNA encapsulation and delivery, which was fabricated with poly(ethylene glycol)-b-poly(d,l-lactide), siRNA and a cationic lipid, using a double emulsion-solvent evaporation technique. By incorporation of the cationic lipid, the encapsulation efficiency of siRNA into the nanoparticles could be above 90% and the siRNA loading weight ratio was up to 4.47%, while the diameter of the nanoparticles was around 170 to 200nm. The siRNA retained its integrity within the nanoparticles, which were effectively internalized by cancer cells and escaped from the endosome, resulting in significant gene knockdown. This effect was demonstrated by significant down-regulation of luciferase expression in HepG2-luciferase cells which stably express luciferase, and suppression of polo-like kinase 1 (Plk1) expression in HepG2 cells, following delivery of specific siRNAs by the nanoparticles. Furthermore, the nanoparticles carrying siRNA targeting the Plk1 gene were found to induce remarkable apoptosis in both HepG2 and MDA-MB-435s cancer cells. Systemic delivery of specific siRNA by nanoparticles significantly inhibited luciferase expression in an orthotopic murine liver cancer model and suppressed tumor growth in a MDA-MB-435s murine xenograft model, suggesting its therapeutic promise in disease treatment.

Targeted delivery of PLK1-siRNA by ScFv suppresses Her2 + breast cancer growth and metastasis

Antibody-mediated delivery of anticancer siRNAs suppresses Her2+ breast cancer growth and metastasis. A Bull’s-Eye for Breast Cancer The goal in archery is to hit the center of the target. Although this could be accomplished by randomly shooting a barrage of arrows, it would be more efficient—and less likely to provoke emergency room visits—to aim straight at the bull’s-eye. Cancer therapies work on a similar principle. Broad therapies may treat the cancer but have many unwanted effects on healthy tissue. Yao et al. now target cancer drugs directly to the tumor using single-chain fragmented antibodies (ScFvs). About 60% of metastatic breast cancers that express human epidermal growth factor receptor 2 (Her2) do not respond to the anti-Her2 therapeutic antibody trastuzumab. The authors hypothesized that ScFvs specific to Her2 could deliver small interfering RNA (siRNA) to Her2+ breast cancer cells. They complexed siRNA for Polo-like kinase 1 (PLK1), which promotes cell division, with a Her2-ScFv-protamine peptide fusion protein (F5-P). This complex suppressed Her2+ breast cancer cell lines and primary human cancers in orthotopic breast cancer models. The siRNA complexes slowed tumor cell growth, reduced metastasis, and prolonged survival with no observed toxicity. The antitumor effects were even greater when a mix of siRNAs was delivered. These results suggest that as a new platform to deliver siRNAs to specifically treat Her2+ breast cancers, F5-P may be on target. A major obstacle to developing small interfering RNAs (siRNAs) as cancer drugs is their intracellular delivery to disseminated cancer cells. Fusion proteins of single-chain fragmented antibodies (ScFvs) and positively charged peptides deliver siRNAs into specific target cells. However, the therapeutic potential of ScFv-mediated siRNA delivery has not been evaluated in cancer. Here, we tested whether Polo-like kinase 1 (PLK1) siRNAs complexed with a Her2-ScFv-protamine peptide fusion protein (F5-P) could suppress Her2+ breast cancer cell lines and primary human cancers in orthotopic breast cancer models. PLK1-siRNAs transferred by F5-P inhibited target gene expression, reduced proliferation, and induced apoptosis of Her2+ breast cancer cell lines and primary human cancer cells in vitro without triggering an interferon response. Intravenously injected F5-P/PLK1-siRNA complexes concentrated in orthotopic Her2+ breast cancer xenografts and persisted for at least 72 hours, leading to suppressed PLK1 gene expression and tumor cell apoptosis. The intravenously injected siRNA complexes retarded Her2+ breast tumor growth, reduced metastasis, and prolonged survival without evident toxicity. F5-P–mediated delivery of a cocktail of PLK1, CCND1, and AKT siRNAs was more effective than an equivalent dose of PLK1-siRNAs alone. These data suggest that F5-P could be used to deliver siRNAs to treat Her2+ breast cancer.

Single-step assembly of cationic lipid-polymer hybrid nanoparticles for systemic delivery of siRNA.

The clinical success of therapeutics of small interfering RNA (siRNA) is still hindered by its delivery systems. Cationic polymer or lipid-based vehicles as the major delivery systems of siRNA cannot sufficiently satisfy siRNA therapeutic applications. It is hypothesized that cationic lipid-polymer hybrid nanoparticles may take advantage of both polymeric and lipid-based nanoparticles for siRNA delivery, while diminishing the shortcomings of both. In this study, cationic lipid-polymer hybrid nanoparticles were prepared by a single-step nanoprecipitation of a cationic lipid (N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl aminoethyl) ammonium bromide, BHEM-Chol) and amphiphilic polymers for systemic delivery of siRNA. The formed hybrid nanoparticles comprised a hydrophobic polylactide core, a hydrophilic poly(ethylene glycol) shell, and a cationic lipid monolayer at the interface of the core and the shell. Such hybrid nanoparticles exhibited excellent stability in serum and showed significantly improved biocompatibility compared to that of pure BHEM-Chol particles. The hybrid nanoparticles were capable of delivering siRNA into BT474 cells and facilitated the escape of loaded siRNA from the endosome into the cytoplasm. The hybrid nanoparticles carrying polo-like kinase 1 (Plk1)-specific siRNA (siPlk1) remarkably and specifically downregulated expression of the oncogene Plk1 and induced cancer cell apoptosis both in vitro and in vivo and significantly suppressed tumor growth following systemic administration. We demonstrate that this system is stable, nontoxic, highly efficient, and easy to scale up, bringing the clinical application of siRNA therapy one important step closer to reality.

Block Copolymer of Polyphosphoester and Poly(l-Lactic Acid) Modified Surface for Enhancing Osteoblast Adhesion, Proliferation, and Function

Surface modification is often needed in tissue engineering to enhance the interaction between cells and synthetic materials and improve the cytocompatibility and cellular functions. In this study, block copolymers of poly(L-lactic acid) and poly(ethyl ethylene phosphate) (PLLA-b-PEEP) were synthesized and used to modify the PLLA surface via a spin-coating process, to understand whether surface modification with polyphosphoester-based polymer will be osteoinductive for potential bone tissue engineering applications. X-ray photoelectron spectra measurements revealed that phosphorus atomic compositions after surface modification increased from 2.09% to 4.39% with increasing PEEP length of PLLA-b-PEEP from 58 to 224 units, which also led to a more hydrophilic surface property compared with unmodified PLLA. The initial osteoblast attachment and proliferation on the modified surfaces were significantly enhanced. Moreover, cellular alkaline phosphatase activity and mineral calcium depositions were also promoted by PEEP modification. The gene expression determined by reverse transcription polymerase chain reaction further revealed that type I collagen and osteocalcin expression were upregulated in osteoblasts cultured on the modified surfaces, indicating that PEEP modification might be potentially osteoinductive and favorable for further application in bone tissue engineering.

Doxorubicin Conjugate of Poly(Ethylene Glycol)‐Block‐Polyphosphoester for Cancer Therapy

Polyphosphoesters with repeating phosphoester linkages in the backbone can be easily functionalized, are biodegradable and potentially biocompatible, and may be potential candidates as polymer carriers of drug conjugates. Here, the efficacy of a polyphosphoester drug conjugate as an anticancer agent in vivo is assessed for the first time. With controlled synthesis, doxorubicin conjugated to poly(ethylene glycol)-block-polyphosphoester (PPEH-DOX) via labile hydrazone bonds form spherical nanoparticles in aqueous solution with an average diameter of ≈60 nm. These nanoparticles are effectively internalized by MDA-MB-231 breast cancer cells and release the conjugated doxorubicin in response to the intracellular pH of endosomes and lysosomes, resulting in significant antiproliferative activity in cancer cells. Compared with free doxorubicin injection, PPEH-DOX injection exhibits much longer circulation behavior in the plasma of mice and leads to enhanced drug accumulation in tumor cells. In an MDA-MB-231 xenograft murine model, inhibition of tumor growth with systemic delivery of PPEH-DOX nanoparticles is more pronounced compared with free doxorubicin injection, suggesting the potential of polyphosphoesters as carriers of drug conjugates in cancer therapy.

Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles.

There is no effective clinical therapy yet for triple-negative breast cancer (TNBC) without particular human epidermal growth factor receptor-2, estrogen and progesterone receptor expression. In this study, we report a molecularly targeted and synthetic lethality-based siRNA therapy for TNBC treatment, using cationic lipid assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the siRNA carrier. It is demonstrated that only in c-Myc overexpressed TNBC cells, while not in normal mammary epithelial cells, delivery of siRNA targeting cyclin-dependent kinase 1 (CDK1) with the nanoparticle carrier (NPsiCDK1) induces cell viability decreasing and cell apoptosis through RNAi-mediated CDK1 expression inhibition, indicating the synthetic lethality between c-Myc with CDK1 in TNBC cells. Moreover, systemic delivery of NPsiCDK1 is able to suppress tumor growth in mice bearing SUM149 and BT549 xenograft and cause no systemic toxicity or activate the innate immune response, suggesting the therapeutic promise with such nanoparticles carrying siCDK1 for c-Myc overexpressed triple negative breast cancer.

Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4.

Anti-Her2 single-chain antibody mediated DNMTs-siRNA delivery for targeted breast cancer therapy.

The targeted delivery of small interfering RNA (siRNA) to specific tumor tissues and tumor cells remains as one of the key challenges in the development of RNA interference as a therapeutic application. To target breast cancer, we developed a therapeutic delivery system using a fusion protein of an anti-Her2 single-chain antibody fragment with a positively charged protamine, namely F5-P, as the carrier to specifically deliver siRNA-targeting DNA methyltransferases 1 and/or 3b genes (siDNMTs) into Her2-expressing breast tumor cells. The carrier F5-P, expressed by the Escherichia coli system, was able to bind siRNA molecules and specifically deliver the siRNA to Her2-expressing BT474 breast cancer cells but not Her2-nonexpressing MDA-MB-231 breast cancer cells, while delivery of siDNMTs to BT474 cells successfully silenced the expression of targeted DNA methyltransferases (DNMTs) and facilitated the de-methylation of the RASSF1A tumor suppressor gene promoter, leading to the suppression of tumor cell proliferation. Moreover, as demonstrated in the BT474 xenograft murine model, F5-P successfully delivered siRNA into a Her2-expressing breast tumor, and tumor growth inhibition was mediated by an intravenous injection of F5-P/siDNMTs complex by down-regulating the expression of DNMTs and restoring tumor suppressor gene expression. These data suggest that the delivery of siDNMTs by F5-P could be used to treat Her2-expressing breast cancer.