Tumor vaccines have demonstrated a modest response rate, primarily attributed to their inefficient delivery to dendritic cells (DCs), low cross-presentation, DC-intrinsic immunosuppressive signals, and an immunosuppressive tumor microenvironment (TME). Here, draining lymph node (DLN)-targeted and tumor-targeted nanovaccines were proposed to address these limitations, and heterocyclic lipidoid (A18) and polyester (BR647) were synthesized to achieve dual-targeted cancer immunotherapy. Meanwhile, oligo hyaluronic acid (HA) and DMG-PEG2000-Mannose were incorporated to prepare dual-targeted nanovaccines encapsulated with STAT3 siRNA and model antigens. The nanovaccines were designed to target the DLN and the tumor, facilitating the delivery of cargo into the cytoplasm. These dual-targeted nanovaccines improved antigen presentation and DC maturation, activated the stimulator of interferon genes (STING) pathway, enhanced the pro-apoptotic effect, and stimulated antitumor immune responses. Additionally, these dual-targeted nanovaccines overcame immunosuppressive TME, reduced immunosuppressive cells, and promoted the polarization of tumor-associated neutrophils from N2 to N1. Among the four dual-targeted nanovaccines that induced robust antitumor responses, the heterocyclic lipidoid@polyester hybrid nanovaccines (MALO@HBNS) demonstrated the most promising results. Furthermore, a combination strategy involving MALO@HBNS and an anti-PD-L1 antibody exhibited an immensely powerful anticancer role. This work introduced a dual-targeted nanovaccine platform for antitumor treatment, suggesting its potential combination with an immune checkpoint blockade as a comprehensive anticancer strategy.

Gene therapy is considered to be a valuable strategy for effective cancer treatment. However, the development of effective delivery systems that can specifically deliver gene materials, such as siRNA to tumor tissues plays a critical role in cancer therapy. In the present study, we have developed a novel complex that is based on an electrostatic interaction between cationic polyurethane ionene (CPUI) nanoparticles and an anti-signal transducer and activator of transcription 3 (STAT3) siRNA. For active targeting, hyaluronic acid (HA) was used to coat the complexes, which significantly reduced the cytotoxicity of the blank nanocarriers while demonstrating high transport efficiency of the siRNA via the CD44-mediated endocytosis pathway in MCF-7 breast cancer cells. The targeted nanocarriers (HA/CPUI/siRNA) showed significantly higher cellular internalization in flow cytometry and confocal microscopy compared with the non-targeted system (CPUI/siRNA). In addition, the incorporation of HA on the surface of the complexes resulted in significantly greater suppression of the STAT3 gene compared to the corresponding non-targeted formulation. Whole-body fluorescence images showed more significant tumor accumulation of the targeted nanocarriers in 4T1 breast tumor-bearing mice. Therefore, HA/CPUI/siRNA nanocarriers are an interesting option for the siRNA-targeted treatment of breast cancer cells.

Co-delivery of anticancer drugs and biologics can provide synergetic effects and outperform single delivery therapies. Here, a nanoparticle (NP) system for co-delivery of methotrexate (MTX) and STAT3 siRNA has been developed and tested in vitro. Mesoporous silica nanoparticles (MSNs) were functionalized with chitosan (ch) by covalent grafting mediated by aminopropyl triethoxysilane (APTES) via glutaraldehyde as the linker. Co-delivery of MTX and STAT3 siRNA to MCF7 cells was demonstrated in cells by flow cytometric analysis and confocal laser scanning fluorescence microscopy for use in breast cancer treatment. MTX either competitively inhibits the dihydrofolate reductase (DHFR) receptor or suppresses the STAT3 metabolic pathway. STAT3 protein plays an essential role in cell division, proliferation and survival. Reduction of the protein by both MTX and STAT3 siRNA, achieved by chMSNs, significantly decreased the viability of breast cancer cells compared to single treatments alone. Cellular uptake of modified NPs was increased over time when additional free MTX was added implicating the DHFR receptor in uptake. In addition, protein corona compositions coated the NPs outer surface, were different between the NPs with and without drug potentially modulating cellular uptake. This study is the first report on co-delivery of MTX and STAT3 siRNA by chitosan modified MSNs.

Objective: In this study, we aimed to develop a candidate modifited polymeric nanoparticle (NP) system that will kill cancer cells by facilitated to apoptosis and also reduce pain. Significance: The primary goal of treatment, especially for metastatic cancers, is to control the growth of the cancer and to alleviate the symptoms. Pain is one of the commonest symptoms of cancer. In cancer treatment, directing cancer cells to death while simultaneously relieving pain will be a new approach. Methods: Chitosan-modified PLGA NPs were prepared using an nanoprecipitation technique. The NPs were loaded with flurbiprofen and decorated with folic acid. STAT3-siRNA was adsorbed to these polymeric NPs using antisense technology. Results: The NPs were small in size (176.9-220.3 nm) with positive zeta potential (+14.1 mV to +27.2 mV). They had high loading capacity and prolonged release properties over 144 hours. Cytotoxicity studies performed with siRNA showed effective electrostatic interaction due to the positively charged NPs. Folic acid facilitated entry into cancer cells and helped to kill them. Conclusion: The formulation we developed is a potential carrier system for both treatment of cancer and prevention of pain, especially for metastatic cancers.

The aim of the present study was to evaluate the effectiveness of iontophoretic co-delivery of curcumin and anti-STAT3 siRNA using cationic liposomes against skin cancer. Curcumin was encapsulated in DOTAP-based cationic liposomes and then complexed with STAT3 siRNA. This nanocomplex was characterized for the average particle size, zeta-potential, and encapsulation efficiency. The cell viability studies in B16F10 mouse melanoma cells have shown that the co-delivery of curcumin and STAT3 siRNA significantly (p < 0.05) inhibited the cancer cell growth compared with either liposomal curcumin or STAT3 siRNA alone. The curcumin-loaded liposomes were able to penetrate up to a depth of 160 μm inside the skin after iontophoretic (0.47 mA/cm2) application. The in vivo efficacy studies were performed in the mouse model of melanoma skin cancer. Co-administration of the curcumin and STAT3 siRNA using liposomes significantly (p < 0.05) inhibited the tumor progression as measured by tumor volume and tumor weight compared with either liposomal curcumin or STAT3 siRNA alone. Furthermore, the iontophoretic administration of curcumin-loaded liposome-siRNA complex showed similar effectiveness in inhibiting tumor progression and STAT3 protein suppression compared with intratumoral administration. Taken together, cationic liposomes can be utilized for topical iontophoretic co-delivery of small molecule and siRNA for effective treatment of skin diseases.

Co-delivery of chemotherapeutic agents improve anti-tumor efficacy and reduce cancer resistance. Here, we report development of layer-by-layer assembled gold nanoparticles (LbL-AuNP) containing anti-STAT3 siRNA and imatinib mesylate (IM) to treat melanoma. The combination treatment with STAT3 siRNA and IM in B16F10 melanoma cells showed greater suppression of STAT3 protein, decreased cell viability and increased apoptotic events compared with LbL-AuNP containing either STAT3 siRNA or IM. In vivo efficacy studies in melanoma tumor bearing mice showed that non-invasive topical iontophoretic administration (0.5mA/cm2) of LbL-AuNP was comparable with intratumoral administration. Co-delivery of STAT3 siRNA and IM using LbL-AuNP showed significant (p<0.05) reduction in percentage tumor volume, tumor weight and suppressed STAT3 protein expression compared with either STAT3 siRNA or IM loaded LbL-AuNP. Taken together, LbL-AuNP can be developed as nanocarrier system for co-delivery of siRNA and small molecule drugs for topical iontophoretic delivery.

Skin cancer is one of the most widely prevalent cancer types with over expression of multiple oncogenic signaling molecules including STAT3. Curcumin is a natural compound with effective anti-cancer properties. The objective of this work was to investigate the liposomal co-delivery of curcumin and STAT3 siRNA by non-invasive topical iontophoretic application to treat skin cancer. Curcumin was encapsulated in cationic liposomes and then complexed with STAT3 siRNA. The liposomal nanocomplex was characterized for particle size, zeta-potential, drug release and stability. Human epidermoid (A431) cancer cells were used to study the cell uptake, growth inhibition and apoptosis induction of curcumin-loaded liposome-siRNA complex. Topical iontophoresis was applied to study the skin penetration of nanocomplex in excised porcine skin model. Results showed that curcumin-loaded liposome-siRNA complex was rapidly taken up by cells preferentially through clathrin-mediated endocytosis pathway. The co-delivery of curcumin and STAT3 siRNA using liposomes resulted in significantly (p < .05) greater cancer cell growth inhibition and apoptosis events compared with neat curcumin and free STAT3 siRNA treatment. Furthermore, topical iontophoresis application enhanced skin penetration of nanocomplex to penetrate viable epidermis. In conclusion, cationic liposomal system can be developed for non-invasive iontophoretic co-delivery of curcumin and siRNA to treat skin cancer.

Overexpression of signal transducer and activator of transcription 3 (STAT3) protein prevents apoptosis and enhances proliferation of melanocytes. The aim of this study was to investigate the feasibility of using layer-by-layer assembled gold nanoparticles (LbL-AuNP) as a carrier for iontophoretic delivery of STAT3 siRNA to treat melanoma. Chitosan coated AuNP (AuNP-CS) were prepared by direct reduction of HAuCl4 in the presence of chitosan. The AuNP-CS were then sequentially layered with siRNA and chitosan to form AuNP-CS/siRNA/CS. STAT3 siRNA replaced with scrambled siRNA or sodium alginate were used as controls. The average particle size and zeta-potential of the prepared LbL-AuNP were 150±10nm (PDI: 0.41±0.06) and 35±6mV, respectively. In vitro studies in B16F10 murine melanoma cells showed that AuNP-CS/siRNA/CS inhibited the cell growth by 49.0±0.6% and 66.0±0.2% at 0.25nM and 0.5nM STAT3 siRNA concentration, respectively. Fluorescence microscopy and flow cytometry studies showed a time dependent cell uptake of the LbL-AuNP up to 120min. Clathrin mediated endocytosis was found to be the predominant cell uptake mechanism for LbL-AuNP. STAT3 siRNA loaded LbL-AuNP reduced the STAT3 protein expression by 47.3% in B16F10 cells. Similarly, apoptosis assay showed 29% and 44% of early and late apoptotic events, respectively after treatment with STAT3 siRNA loaded LbL-AuNP. Confocal microscope and skin cryosections showed that application of 0.47mA/cm(2) of anodal iontophoresis enhanced the skin penetration of LbL-AuNP to reach viable epidermis. In conclusion, layer-by-layer chitosan coated AuNP can be developed as a carrier for iontophoretic delivery of STAT3 siRNA to treat melanoma.

OBJECTIVE: To investigate the effects of lentivirus mediated STAT3 silencing on human chronic myeloid leukemia cells (K562) and the growth of chronic myeloid leukemia mice as well as to explore the potential mechanisms.
METHODS: Unbtreated K562 cells (CON), blank lentivirus transfected K562 cells (NC) and K562 cells expressing STAT3 siRNA (STAT3 siRNA) were injected into SCID mice to establish the chronic myeloid leukemia model in mice. The growth, peripheral white blood cell count and spleen index in these mice were determined.
RESULTS: In vitro experiment showed, when compared with control group, the interference efficiency of STAT3 expression was as high as 97.5% in K562 cells. Western blot assay revealed that the expression of c-Myc, Bcl-xL and Cyclin D1 reduced by 17.01%, 7.3% and 6.82%, respectively, showing significant difference when compared with control group (P < 0.01). These findings were consistent with those from fluorescence quantitative PCR. In vivo experiment showed the body weight of mice reduced progressively and the peripheral white blood cell count increased gradually in control group, accompanied by dragging hind limbs and progressive enlargement of the spleen. The body weight remained unchanged, peripheral white blood cell count reduced gradually and the spleen did not enlarge in mice treated with STAT3 siRNA expressing cells.
CONCLUSIONS: Lentivirus mediated STAT3 silencing may inhibit the expression of its downstream genes (c-Myc, Bcl-xL and Cyclin D1) related to cell proliferation, apoptosis and cycle to suppress the malignant biological behaviors, and STAT3 silencing also inhibit the leukemogenic potency of K562 cells in mice.

Although cancer vaccine-based immunotherapy holds great potential for cancer treatment, tumor-induced dendritic cell (DC) dysfunction remains to be the major obstacle for developing effective vaccines. Compared with normal DCs, tumor-associated DCs (TADCs) are less matured with poor responsiveness to Toll-like receptor (TLR) stimulation, which has been related with STAT3 hyperactivity. In the present study, Poly I:C (PIC, a TLR3 agonist), STAT3 siRNA and OVA antigen were co-encapsulated by poly (ethylene glycol)-b-poly (L-lysine)-b-poly (L-leucine) (PEG-PLL-PLLeu) polypeptide micelles to generate PMP/OVA/siRNA nanovaccine, which was aimed to effectively overcome DC dysfunction in vivo by deleting STAT3 gene in situ. The results showed that PMP/OVA/siRNA simultaneously facilitated the cellular uptake of OVA antigen and siRNA about 3-200 folds, and decreased STAT3 expression in TADCs over 50% both in vitro and in vivo. PMP/OVA/siRNA also elevated CD86 and CD40 expression as well as IL-12 production by TADCs more effectively than PMP/OVA did, indicating its strong potency of inducing TADC maturation and activation. Moreover, the immunization of PMP/OVA/siRNA rather than PMP/OVA effectively abrogated immunosuppression in the tumor microenvironment by increasing mature DCs and decreasing immunosuppressive cells in tumor-draining lymph nodes, which thereby led to potent anti-tumor immune responses and dramatic tumor regression with prolonged survival. Hence, in vivo co-delivery of immunopotentiator (PIC) and immunosuppressive gene silencer (STAT3 siRNA) by nanovaccines are expected to be a promising strategy to improve the therapeutic efficacy of cancer vaccines by modulating TADCs and overcoming tumor immunosupression.