Mitigation of Anti-Drug Antibody Production for Augmenting Anticancer Efficacy of Therapeutic Protein via Co-Injection of Nano-Rapamycin.

The induction of anti-drug antibody (ADA) is a formidable challenge for protein-based therapy. Trichosanthin (TCS) as a class of ribosome-inactivating proteins is widely studied in tumor treatment. However, the immunogenicity can induce the formation of ADA, which can cause hypersensitivity reactions and neutralize the efficacy of TCS, thus limiting its clinical application in cancer therapy. Here, a promising solution to this issue is presented by co-administration of the rapamycin nanoparticles and TCS. PEGylated rapamycin amphiphilic molecule is designed and synthesized as a prodrug and a delivery carrier, which can self-assemble into a nanoparticle system with encapsulation of free rapamycin, a hydrophobic drug. It is found that co-injection of the PEGylated rapamycin nanoparticles and TCS could mitigate the formation of anti-TCS antibody via inducing durable immunological tolerance. Importantly, the combination of TCS and the rapamycin nanoparticles has an enhanced effect on inhibit the growth of breast cancer. This work provides a promising approach for protein toxin-based anticancer therapy and for promoting the clinical translation.

cRGD-modified nanoparticles of multi-bioactive agent conjugate with pH-sensitive linkers and PD-L1 antagonist for integrative collaborative treatment of breast cancer.

Targeted co-delivery and co-release of multi-drugs is essential to have an integrative collaborative effect on treating cancer. It is valuable to use few drug carriers for multi-drug delivery. Herein, we develop cRGD-modified nanoparticles (cRGD-TDA) of a conjugate of doxorubicin as cytotoxic agent, adjudin as an anti-metastasis agent and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) as a reactive oxygen species inducer linked with pH-sensitive bonds, and then combine the nanoparticles with PD-L1 antagonist to treat 4T1 triple-negative breast cancer. cRGD-TDA NPs present tumor-targeted co-delivery and pH-sensitive co-release of triple agents. cRGD-TDA NPs combined with PD-L1 antagonist much more significantly inhibit tumor growth and metastasis than single-drug treatment, which is due to their integrative collaborative effect. It is found that TPGS elicits a powerful immunogenic cell death effect. Meanwhile, PD-L1 antagonist mitigates the immunosuppressive environment and has a synergistic effect with the cRGD-TDA NPs. The study provides a new strategy to treat refractory cancer integratively and collaboratively.

Paclitaxel-loaded ROS-responsive nanoparticles for head and neck cancer therapy.

High intracellular reactive oxygen species (ROS) level is characteristic of cancer cells and could act as a target for the efficient targeted drug delivery for cancer treatment. Consequently, biomaterials that react to excessive levels of ROS are essential for biomedical applications. In this study, a novel ROS-responsive polymer based on D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) and poly (ß-thioester) (TPGS-PBTE) was synthesized for targeted delivery of the first-line antineoplastic drug, paclitaxel (PTX). The resultant TPGS-PBTE NPs showed good ROS-responsive capability in size change and drug release. Compared to PTX, PTX-loaded nanoparticles (PTX@TPGS-PBTE NPs) showed enhanced cytotoxicity and higher level of apoptosis toward squamous cell carcinoma (SCC-7) cells. Tumor-targeted delivery of the NPs was also observed, especially after being modified with a tumor-targeting peptide, cRGD. Enhanced tumor growth inhibition was also observed in head and neck cancer SCC-7 murine models. In summary, PTX@TPGS-PBTE NPs can achieve good therapeutic effects of PTX against head and neck cancer both in vitro and in vivo, especially when modified by cRGD for active targeting, which enriched the application of ROS responsive system utilized in the delivery of anticancer drugs.

Cisplatin and oleanolic acid Co-loaded pH-sensitive CaCO3 nanoparticles for synergistic chemotherapy.

Despite being one of the most potent anticancer agents, cisplatin (CDDP) clinical usage is limited owing to the acquired resistance and severe adverse effects including nephrotoxicity. The current work has offered a unique approach by designing a pH-sensitive calcium carbonate drug delivery system for CDDP and oleanolic acid (OA) co-delivery, with an enhanced tumor efficacy and reduced unwanted effects. Micro emulsion method was employed to generate calcium carbonate cores (CDDP encapsulated) followed by lipid coating along with the OA loading resulting in the generation of lipid-coated cisplatin/oleanolic acid calcium carbonate nanoparticles (CDDP/OA-LCC NPs). In vitro biological assays confirmed the synergistic apoptotic effect of CDDP and OA against HepG2 cells. It was further verified in vivo through the tumor-bearing nude mice model where NPs exhibited enhanced satisfactory antitumor efficacy in contrast to free drug solutions. In vivo pharmacokinetic study demonstrated that a remarkable long circulation time with a constant therapeutic concentration for both drugs could be achieved via this drug delivery system. In addition, the in vivo imaging study revealed that DiR-loaded NPs were concentrated more in tumors for a longer period of time as compared to other peritoneal tissues in tumor bearing mice, demonstrating the site specificity of the delivery system. On the other hand, hematoxylin and eosin (H&E) staining of Kunming mice kidney tissue sections revealed that OA greatly reduced CDDP induced nephrotoxicity in the formulation. Overall, these results confirmed that our pH-sensitive dual loaded drug delivery system offers a handy direction for effective and safer combination chemotherapy.

TMTP1-Modified, Tumor Microenvironment Responsive Nanoparticles Co-Deliver Cisplatin and Paclitaxel Prodrugs for Effective Cervical Cancer Therapy.

BACKGROUND AND PURPOSE: Cisplatin-paclitaxel (TP) combination chemotherapy as the first-line therapy for numerous cancers is hindered by its inadequate accumulation in tumors and severe side effects resulting from non-specific distribution. The aim of this study is to explore whether TMTP1-modified, cisplatin and paclitaxel prodrugs co-loaded nanodrug could improve cervical cancer chemotherapy and relieve its side effects through active and passive tumor targeting accumulation and controlled drug release. METHODS: TDNP, with capacities of active targeting for tumors and controlled drug release, was prepared to co-deliver cisplatin and paclitaxel prodrugs. The characteristics were investigated, including the diameter, surface zeta potential, stability and tumor microenvironment (TME) dependent drug release profiles. Cellular uptake, cytotoxicity, drug accumulation in tumors, antitumor effects and safety analysis were evaluated in vitro and in vivo. RESULTS: The oxidized cisplatin and the paclitaxel linked to the polymer achieved a high loading effciency of over 80% and TME-dependent sustained drug release. Moreover, TMTP1 modification enhanced cellular uptake of TDNP and further improved the cytotoxicity of TDNP in vitro. In vivo, TDNP showed an extended blood circulation and increased accumulation in SiHa xenograft models with the aid of TMTP1. More importantly, TDNP controlled tumor growth without life-threatening side effects. CONCLUSION: Our study provided a novel TP co-delivery platform for targeted chemotherapy of cervical cancer, which was promising to improve the therapeutic effcacy of TP and may also have application in other tumors.

An effective vaginal gel to deliver CRISPR/Cas9 system encapsulated in poly (ß-amino ester) nanoparticles for vaginal gene therapy.

BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (ß-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.

Oxymatrine Liposomes for Intervertebral Disc Treatment: Formulation, in vitro and vivo Assessments.

PURPOSE: Intervertebral disc degeneration (IVDD) is the main cause of modern low back pain, leading to high societal economic costs. To find an effective medical treatment for this disease, oxymatrine liposomes (OMT-LIP) were prepared with the pH-gradient method. MATERIALS AND METHODS: Nucleus pulposus (NP) cells from Sprague-Dawley rats were used for the cell experiments. Kunming mice were used for in vivo imaging. LIP were employed to deliver OMT, and the particle size, ζ-potential, morphology, in vitro stability and in vitro release characteristics were evaluated. The OMT-LIP targeting effect was measured by in vivo imaging. Cell Counting Kit-8 assays were used to detect the cytotoxicity of OMT and OMT-LIP on NP cells. Therapeutic efficacy was measured by Western blot, real-time quantitative polymerase chain reaction, and apoptosis assays. Radiologic analysis was performed to evaluate the therapeutic effects in vivo. RESULTS: Orthogonal test results revealed that the mass ratio of egg yolk phosphatidylcholine to cholesterol was the key factor to effectively trap OMT in LIP. Optimal OMT-LIP showed multivesicular structure with entrapment efficiency of 73.4 ± 4.1%, particle size of 178.1 ± 2.9 nm, and ζ-potential of -13.30 ± 2.34 mV. OMT-LIP manifested excellent stability in vitro and presented significantly longer sustained release compared to OMT solution in phosphate-buffered saline (pH 7.4). OMT-LIP conspicuously increased OMT accumulation in the degenerative disc, attenuated NP cell apoptosis, reduced the expression of matrix metalloproteinases 3/9 and interleukin-6, and decreased degradation of type II collagen. In in vivo study, X-ray demonstrated that OMT-LIP inhibited IVDD. CONCLUSION: OMT-LIP may be a useful treatment to alleviate disc inflammation and IVDD.

Hyperbranched poly(ß-amino ester) based polyplex nanopaticles for delivery of CRISPR/Cas9 system and treatment of HPV infection associated cervical cancer.

Persistent high-risk HPV infection is the main factor for cervical cancer. HPV E7 oncogene plays an important role in HPV carcinogenesis. Down-regulation of E7 oncogene expression could induce growth inhibition in HPV-positive cells and thus treats HPV related cervical cancer. Here we developed a non-virus gene vector based on poly(amide-amine)-poly(ß-amino ester) hyperbranched copolymer (hPPC) for the delivery of CRISPR/Cas9 system to specifically cleave HPV E7 oncogene in HPV-positive cervical cancer cells. The diameter of polyplex nanoparticles (NPs) formed by hPPCs/linear poly(ß-amino ester) (PBAE) and plasmids were approximately 300 nm. These hPPCs/PBAE-green fluorescence protein plasmids polyplex NPs showed high transfection efficiency and low toxicity in cells and mouse organs. By cleaving HPV16 E7 oncogene, reducing the expression of HPV16 E7 protein and increasing intracellular retinoblastoma 1 (RB1) amount, hPPCs/PBAE-CRISPR/Cas9 therapeutic plasmids polyplex NPs, especially highly branched hPPC1-plasmids polyplex NPs, exhibited strong growth inhibition of cervical cancer cells in vitro and xenograft tumors in nude mice. Together, the hPPCs/PBAE polyplex NPs to deliver HPV16 E7 targeted CRISPR/Cas9 system in this study could potentially be applied to treat HPV-related cervical cancer.

Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity.

Blocking the programmed death-ligand 1 (PD-L1) on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy. However, only a minority of patients presented immune responses in clinical trials. To develop an alternative treatment method based on immune checkpoint blockade, we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5 (Cdk5) gene in vivo. The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5, leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer. Importantly, we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8+ T cells was significantly increased while regulatory T cells (Tregs) was decreased. It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy. It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.

TMTP1-modified Indocyanine Green-loaded Polymeric Micelles for Targeted Imaging of Cervical Cancer and Metastasis Sentinel Lymph Node in vivo.

Metastasis is one of the most threatening aspects of cervical cancer. We developed a method to intraoperatively map the primary tumor, metastasis and metastatic sentinel lymph nodes (SLNs), providing real-time intraoperative guidance in cervical cancer. Methods: TMTP1, a tumor metastasis targeting peptide, was employed to modify the indocyanine green (ICG)-loaded poly (ethylene glycol)- poly (lactic-co-glycolic acid) (PEG-PLGA) micelles. The cervical cancer subcutaneous tumor model and lung metastasis model were established to determine the active targeting of ICG-loaded TMTP1-PEG-PLGA micelles (ITM) for the primary tumor and occult metastasis of cervical cancer. Human cervical cancer HeLa cells engineered by firefly luciferase were injected into the right hocks of BALB/c nude mice to develop the SLN metastasis model. The ITM and control ICG-loaded PEG-PLGA micelles (IM) were injected into the right hind footpads in the SLN metastasis model, and the migration and retention of micelles were recorded under near-infrared fluorescence. K14-HPV16 transgenic mice were also used to detect the image capability of ITM to target cancerous lesions. Results: ITM could actively target imaging of the primary tumor and cervical cancer metastasis. ITM quickly diffused from the injection site to SLNs along lymphatic capillaries and remained in the SLNs for 12 h. Moreover, ITM specifically accumulated in the tumor metastatic SLNs (T-SLNs), which could be successfully distinguished from normal SLNs (N-SLNs). Conclusion: ITM could achieve active targeting of the primary tumor, metastasis and T-SLNs, providing precise and real-time intraoperative guidance for cervical cancer.

Targeted co-delivery of Trp-2 polypeptide and monophosphoryl lipid A by pH-sensitive poly (ß-amino ester) nano-vaccines for melanoma.

Dendritic cell (DC)-targeted vaccines based on nanotechnology are a promising strategy to efficiently induce potent immune responses. We synthesized and manufactured a mannose-modified poly (ß-amino ester) (PBAE) nano-vaccines with easily tuneable and pH-sensitive characteristics to co-deliver the tumor-associated antigen polypeptide Trp-2 and the TLR4 agonist monophosphoryl lipid A (MPLA). To reduce immunosuppression in the tumor microenvironment, an immune checkpoint inhibitor, PD-L1 antagonist, was administrated along with PBAE nano-vaccines to delay melanoma development. We found that mannosylated Trp-2 and MPLA-loaded PBAE nano-vaccines can target and mature DCs, consequently boosting antigen-specific cytotoxic T lymphocyte activity against melanoma. The prophylactic study indicates that combination therapy with PD-L1 antagonist further enhanced anti-tumor efficacy by 3.7-fold and prolonged median survival time by 1.6-fold more than free Trp-2/MPLA inoculation. DC-targeting PBAE polymers have a great potential as a nanotechnology platform to design vaccines and achieve synergistic anti-tumor effects with immune checkpoint therapy.

Doxorubicin and adjudin co-loaded pH-sensitive nanoparticles for the treatment of drug-resistant cancer.

Multi-drug resistance (MDR) of tumor is a major cause of chemotherapy failure. In this study, a pH-sensitive graft copolymer, poly(ß-amino ester)-g-ß-cyclodextrin (PBAE-g-ß-CD), was synthesized via Michael addition polymerization and was employed to co-deliver doxorubicin (DOX), a chemotherapy agent, and adjudin (ADD), a mitochondrial inhibitor, in the form of dual-drug co-loaded nanoparticles (NPs). Specifically, DOX was conjugated to 1-adamantaneacetic acid (Aa) to generate a prodrug that was subsequently encapsulated in the cavity of cyclodextrin via host-guest interactions. In addition, ADD was encapsulated by poly(ß-aminoester) (PBAE). The introduction of the Aa-d-α-tocopheryl polyethylene glycolsuccinate (TPGS) conjugate enhanced the biocompatibility and serum stability of the resulting NPs. The NPs can realize precise ratiometric control of drugs being loaded, increase cellular uptake of the drugs, induce mitochondrial dysfunction and augment tumor treatment efficiency by inducing apoptosis. Western blot and polymerase chain reaction analyses showed that inhibition of P-glycoprotein and X-linked inhibitor of apoptosis protein expression may underlie inhibition of tumor resistance mediated by NPs. The MCF-7/ADR xenograft tumor model also revealed that in comparison with DOX, the NPs exhibited satisfactory performance in promoting apoptosis of tumor cells and achieved high therapeutic outcomes for MDR tumors. STATEMENT OF SIGNIFICANCE: Combination chemotherapy is an effective way to overcome MDR of tumor. However, one of the major obstacles for successful combination chemotherapy is the co-loading, co-delivery and controlled release of two different drugs, whose chemo-physical properties may be totally different. In this study, a pH-sensitive NP system was designed to realize the co-loading and precise ratiometric control of DOX and ADD, as well as the programmed drug release. That is, ADD release was triggered by low pH in endo/lysosome after endocytosis and then DOX was hydrolyzed to achieve a sustained release in tumor cells. Therefore, the NPs exhibited an effectively growth inhibition against MDR cells both in vitro and in vivo via the synergistic effect of ADD and DOX, which provided a promising strategy for treatment of MDR cancer.

Recent developments in d-α-tocopheryl polyethylene glycol-succinate-based nanomedicine for cancer therapy.

Cancer remains an obstacle to be surmounted by humans. As an FDA-approved biocompatible drug excipient, d-α-tocopheryl polyethylene glycol succinate (TPGS) has been widely applied in drug delivery system (DDS). Along with in-depth analyses of TPGS-based DDS, increasingly attractive results have revealed that TPGS is able to act not only as a simple drug carrier but also as an assistant molecule with various bio-functions to improve anticancer efficacy. In this review, recent advances in TPGS-based DDS are summarized. TPGS can inhibit P-glycoprotein, enhance drug absorption, induce mitochondrial-associated apoptosis or other apoptotic pathways, promote drug penetration and tumor accumulation, and even inhibit tumor metastasis. As a result, many formulations, by using original TPGS, TPGS-drug conjugates or TPGS copolymers, were prepared, and as expected, an enhanced therapeutic effect was achieved in different tumor models, especially in multidrug resistant and metastatic tumors. Although the mechanisms by which TPGS participates in such functions are not yet very clear, considering its effectiveness in tumor treatment, TPGS-based DDS appears to be one of the best candidates for future clinical applications.