Tumor targeting peptide TMTP1 modified Antigen capture Nano-vaccine combined with chemotherapy and PD-L1 blockade effectively inhibits growth of ovarian cancer.

The mortality of ovarian cancer (OC) has long been the highest among gynecological malignancies. Although OC is considered to be an immunogenic tumor, the effect of immunotherapy is not satisfactory. The immunosuppressive microenvironment is one reason for this, and the absence of recognized effective antigens for vaccines is another. Chemotherapy, as one of the most commonly used treatment for OC, can produce chemotherapy-associated antigens (CAAs) during treatment and show the effect of in situ vaccine. Herein, we designed an antigen capture nano-vaccine NP-TP1@M-M with tumor targeting peptide TMTP1 and dendritic cell (DC) receptor mannose assembled on the surface and adjuvant monophosphoryl lipid A (MPLA) encapsulated in the core of poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles. PLGA itself possessed the ability of antigen capture. TMTP1 was a tumor-homing peptide screened by our research team, which held extensive and excellent tumor targeting ability. After these modifications, NP-TP1@M-M could capture and enrich more tumor-specific antigens after chemotherapy, stimulate DC maturation, activate the adaptive immunity and combined with immune checkpoint blockade to maximize the release of the body's immune potential, providing an eutherapeutic strategy for the treatment of OC.

Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety.

INTRODUCTION: CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.). OBJECTIVES: To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro. METHODS: We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis. RESULTS: The particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %-40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein-Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.

Post cross-linked ROS-responsive poly(ß-amino ester)-plasmid polyplex NPs for gene therapy of EBV-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common tumors in South China and Southeast Asia and is thought to be associated with Epstein-Barr virus (EBV) infection. Downregulation of latent membrane protein 1 (LMP1) encoded by EBV can reduce the expression of NF-κB and PI3K, induce apoptosis, and inhibit the growth of EBV-related NPC. For targeted cleavage of the Lmp1 oncogene via the CRISPR/Cas9 gene editing system, a post cross-linked ROS-responsive poly(ß-amino ester) (PBAE) polymeric vector was developed for the delivery of CRISPR/Cas9 plasmids both in vitro and in vivo. After composition optimization, the resultant polymer-plasmid polyplex nanoparticles (NPs) showed a diameter of ∼230 nm and a zeta potential of 22.3 mV with good stability. Compared with the non-cross-linked system, the cross-linked NPs exhibited efficient and quick cell uptake, higher transfection efficiency in EBV-positive C666-1 cells (53.5% vs. 40.6%), more efficient gene editing ability against the Mucin2 model gene (Muc2) (17.9% vs. 15.4%) and Lmp1 (8.5% vs. 5.6%), and lower intracellular reactive oxygen species (ROS) levels. The NPs achieved good tumor penetration and tumor growth inhibition in the C666-1 xenograft tumor model via Lmp1 cleavage, indicating their potential for gene therapy of EBV-related NPC.

TPGS-b-PBAE Copolymer-Based Polyplex Nanoparticles for Gene Delivery and Transfection In Vivo and In Vitro.

Poly (ß-amino ester) (PBAE) is an exceptional non-viral vector that is widely used in gene delivery, owing to its exceptional biocompatibility, easy synthesis, and cost-effectiveness. However, it carries a high surface positive charge that may cause cytotoxicity. Therefore, hydrophilic d-α-tocopherol polyethylene glycol succinate (TPGS) was copolymerised with PBAE to increase the biocompatibility and to decrease the potential cytotoxicity of the cationic polymer-DNA plasmid polyplex nanoparticles (NPs) formed through electrostatic forces between the polymer and DNA. TPGS-b-PBAE (TBP) copolymers with varying feeding molar ratios were synthesised to obtain products of different molecular weights. Their gene transfection efficiency was subsequently evaluated in HEK 293T cells using green fluorescent protein plasmid (GFP) as the model because free GFP is unable to easily pass through the cell membrane and then express as a protein. The particle size, ζ-potential, and morphology of the TBP2-GFP polyplex NPs were characterised, and plasmid incorporation was confirmed through gel retardation assays. The TBP2-GFP polyplex NPs effectively transfected multiple cells with low cytotoxicity, including HEK 293T, HeLa, Me180, SiHa, SCC-7 and C666-1 cells. We constructed a MUC2 (Mucin2)-targeting CRISPR/cas9 gene editing system in HEK 293T cells, with gene disruption supported by oligodeoxynucleotide (ODN) insertion in vitro. Additionally, we developed an LMP1 (latent membrane protein 1)-targeting CRISPR/cas9 gene editing system in LMP1-overexpressing SCC7 cells, which was designed to cleave fragments expressing the LMP1 protein (related to Epstein-Barr virus infection) and thus to inhibit the growth of the cells in vivo. As evidenced by in vitro and in vivo experiments, this system has great potential for gene therapy applications.

Chitosan-Functionalized Poly(ß-Amino Ester) Hybrid System for Gene Delivery in Vaginal Mucosal Epithelial Cells.

Gene therapy displays great promise in the treatment of cervical cancer. The occurrence of cervical cancer is highly related to persistent human papilloma virus (HPV) infection. The HPV oncogene can be cleaved via gene editing technology to eliminate carcinogenic elements. However, the successful application of the gene therapy method depends on effective gene delivery into the vagina. To improve mucosal penetration and adhesion ability, quaternized chitosan was introduced into the poly(ß-amino ester) (PBAE) gene-delivery system in the form of quaternized chitosan-g-PBAE (QCP). At a mass ratio of PBAE:QCP of 2:1, the polymers exhibited the highest green fluorescent protein (GFP) transfection efficiency in HEK293T and ME180 cells, which was 1.1 and 5.4 times higher than that of PEI 25 kD. At this mass ratio, PBAE-QCP effectively compressed the GFP into spherical polyplex nanoparticles (PQ-GFP NPs) with a diameter of 255.5 nm. In vivo results indicated that owing to the mucopenetration and adhesion capability of quaternized CS, the GFP transfection efficiency of the PBAE-QCP hybrid system was considerably higher than those of PBAE and PEI 25 kD in the vaginal epithelial cells of Sprague-Dawley rats. Furthermore, the new system demonstrated low toxicity and good safety, laying an effective foundation for its further application in gene therapy.

A TMVP1-modified near-infrared nanoprobe: molecular imaging for tumor metastasis in sentinel lymph node and targeted enhanced photothermal therapy.

BACKGROUND: TMVP1 is a novel tumor targeting polypeptide screened by our laboratory with a core sequence of five amino acids LARGR. It specially binds to vascular endothelial growth factor receptor-3 (VEGFR-3), which is mainly expressed on neo-lymphatic vessels in sentinel lymph node (SLN) with tumor metastasis in adults. Here, we prepared a targeted nanoprobe using TMVP1-modified nanomaterials for tumor metastasis SLN imaging. RESULTS: In this study, TMVP1-modified polymer nanomaterials were loaded with the near-infrared (NIR) fluorescent dye, indocyanine green (ICG), to prepare a molecular imaging TMVP1-ICG nanoparticles (NPs) to identify tumor metastasis in SLN at molecular level. TMVP1-ICG-NPs were successfully prepared using the nano-precipitation method. The particle diameter, morphology, drug encapsulation efficiency, UV absorption spectrum, cytotoxicity, safety, and pharmacokinetic properties were determined. The TMVP1-ICG-NPs had a diameter of approximately 130 nm and an ICG loading rate of 70%. In vitro cell experiments and in vivo mouse experiments confirmed that TMVP1-ICG-NPs have good targeting ability to tumors in situ and to SLN with tumor metastasis by binding to VEGFR-3. Effective photothermal therapy (PTT) with TMVP1-ICG-NPs was confirmed in vitro and in vivo. As expected, TMVP1-ICG-NPs improved ICG blood stability, targeted tumor metastasis to SLN, and enhanced PTT/photodynamic (PDT) therapy, without obvious cytotoxicity, making it a promising theranostic nanomedicine. CONCLUSION: TMVP1-ICG-NPs identified SLN with tumor metastasis and were used to perform imaging-guided PTT, which makes it a promising strategy for providing real-time NIR fluorescence imaging and intraoperative PTT for patients with SLN metastasis.

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.

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.

Enhanced migration and immunoregulatory capacity of BMSCs mediated by overexpression of CXCR4 and IL-35.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been widely studied for their applications in immunoregulation and tissue repair. However, the therapeutic effects of BMSCs in the body are limited, partly due to the low homing efficiency of BMSCs to affected parts. The stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis is well known to play an essential role in the homing of BMSCs. Interleukin 35 (IL-35) is a newly discovered cytokine confirmed to inhibit overactivated immune function and have a good therapeutic effect on autoimmune diseases. In this study, we innovatively developed dual gene modification of BMSCs by transducing CXCR4 and IL-35 and found that the migration and immunomodulatory activity of genetically engineered BMSCs were significantly enhanced compared to their natural counterparts. These results suggest that BMSCs modified by dual overexpression of CXCR4 and IL-35 may provide a potential treatment strategy for autoimmune diseases.

Enhanced photodynamic therapy/photothermo therapy for nasopharyngeal carcinoma via a tumour microenvironment-responsive self-oxygenated drug delivery system.

The hypoxic nature of tumours limits the efficiency of oxygen-dependent photodynamic therapy (PDT). Hence, in this study, indocyanine green (ICG)-loaded lipid-coated zinc peroxide (ZnO2) nanoparticles (ZnO2@Lip-ICG) was constructed to realize tumour microenvironment (TME)-responsive self-oxygen supply. Near infrared light irradiation (808 nm), the lipid outer layer of ICG acquires sufficient energy to produce heat, thereby elevating the localised temperature, which results in accelerated ZnO2 release and apoptosis of tumour cells. The ZnO2 rapidly generates O2 in the TME (pH 6.5), which alleviates tumour hypoxia and then enhances the PDT effect of ICG. These results demonstrate that ZnO2@Lip-ICG NPs display good oxygen self-supported properties and outstanding PDT/PTT characteristics, and thus, achieve good tumour proliferation suppression.

ROS-responsive nanoparticles for oral delivery of luteolin and targeted therapy of ulcerative colitis by regulating pathological microenvironment.

Oxidative stress, caused by excessive production of reactive oxygen species (ROS), plays a crucial role in the occurrence and development of ulcerative colitis (UC). We developed ROS-responsive nanoparticles (NPs) as an efficacious nanomedicine against UC with oral administration. The NPs were fabricated with a d-α-tocopherol polyethylene glycol succinate-b-poly(ß-thioester) copolymer (TPGS-PBTE) for ROS cleavage via the colitis-targeted delivery of luteolin (LUT), a natural flavonoid with good anti-inflammation and radical-scavenging activity. Owing to the thioether bond in the polymer main chain, the TPGS-PBTE NPs exhibited an ROS-responsive size change and drug release, which benefited the ROS-scavenging and selective accumulation of LUT in the inflamed colon. In a dextran sulfate sodium-induced acute colitis murine model, LUT@TPGS-PBTE NPs alleviated body weight loss, colon length shortening, and damage to the colonic tissues due to the suppression of ROS and proinflammatory cytokines (e.g., IL-17A, IL-6, interferon-γ, tumor necrosis factor-α), as well as upregulation of glutathione and anti-inflammatory factors (e.g., IL-10, IL-4). More importantly, LUT@TPGS-PBTE NPs regulated the inflammatory microenvironment by modulating the T helper (Th)1/Th2 and Th17/regulatory T cell (Treg) balance (i.e., increased numbers of Tregs and Th2 cells and decreased numbers of Th1 and Th17 â€‹cells), thus resolving inflammation and accelerating the healing of the intestinal mucosa. Additionally, the LUT@TPGS-PBTE NPs formulation enabled the reduction of the effective dose of LUT and showed excellent biosafety in the mouse model, demonstrating its potential as a targeted UC therapeutic oral preparation.

E7-Targeted Nanotherapeutics for Key HPV Afflicted Cervical Lesions by Employing CRISPR/Cas9 and Poly (Beta-Amino Ester).

INTRODUCTION: Persistent HR-HPV (high-risk human papillomavirus) infection is the main cause of cervical cancer. The HPV oncogene E7 plays a key role in HPV tumorigenesis. At present, HPV preventive vaccines are not effective for patients who already have a cervical disease, and implementation of the recommended regular cervical screening is difficult in countries and regions lacking medical resources. Therefore, patients need medications to treat existing HPV infections and thus block the progression of cervical disease. METHODS: In this study, we developed nanoparticles (NPs) composed of the non-viral vector PBAE546 and a CRISPR/Cas9 recombinant plasmid targeting HPV16 E7 as a vaginal treatment for HPV infection and related cervical malignancies. RESULTS: Our NPs showed low toxicity and high biological safety both in vitro (cell line viability) and in vivo (various important organs of mice). Our NPs significantly inhibited the growth of xenograft tumors derived from cervical cancer cell lines in nude mice and significantly reversed the cervical epithelial malignant phenotype of HPV16 transgenic mice. CONCLUSION: Our NPs have great potential to be developed as a drug for the treatment of HPV-related cervical cancer and precancerous lesions.

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.

DeepEBV: a deep learning model to predict Epstein-Barr virus (EBV) integration sites.

MOTIVATION: Epstein-Barr virus (EBV) is one of the most prevalent DNA oncogenic viruses. The integration of EBV into the host genome has been reported to play an important role in cancer development. The preference of EBV integration showed strong dependence on the local genomic environment, which enables the prediction of EBV integration sites. RESULTS: An attention-based deep learning model, DeepEBV, was developed to predict EBV integration sites by learning local genomic features automatically. First, DeepEBV was trained and tested using the data from the dsVIS database. The results showed that DeepEBV with EBV integration sequences plus Repeat peaks and 2-fold data augmentation performed the best on the training dataset. Furthermore, the performance of the model was validated in an independent dataset. In addition, the motifs of DNA-binding proteins could influence the selection preference of viral insertional mutagenesis. Furthermore, the results showed that DeepEBV can predict EBV integration hotspot genes accurately. In summary, DeepEBV is a robust, accurate and explainable deep learning model, providing novel insights into EBV integration preferences and mechanisms. AVAILABILITYAND IMPLEMENTATION: DeepEBV is available as open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepEBV.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Oxymatrine loaded nitric oxide-releasing liposomes for the treatment of ulcerative colitis.

Oxymatrine (OM) is the biologically active ingredient of Chinese medicinal herb Sophora flavescens, which is reported to be effective on alleviating ulcerative colitis (UC) due to its anti-inflammatory property. However, its highly effective dose is an obstacles to its application. Therefore, liposome was used to encapsulate OM, realize targeting delivery to colitis and thus reduce drug dosage. Meanwhile, considering the potential anti-inflammatory ability of nitric oxide (NO), a NO donor, d-α-tocopheryl polyethylene glycol succinate nitrate (TN), was introduced into the liposomal system and OM loaded NO-releasing liposomes (OM@TN-lip) were prepared in order to co-deliver OM and NO to the inflammatory lesions of DSS-induced UC mice to achieve the combination therapy. OM@TN-lip was multilamelar sphere with the encapsulation efficiency of ~70%, the diameter of ~200 nm and ζ-potential of about -13 mV. Bio-distribution results revealed the liposomes could efficiently accumulate in the inflammatory colon by diffusion and maintain for more than 36 h. In UC mice model, OM@TN-lip showed significant alleviation of inflammation and the treatment was highly related to down-regulation of pro-inflammatory cytokines TNF-α, IFN-γ, IL-1ß and IL-6, decrease of macrophages infiltration, activity decrease of myeloperoxidase (MPO) and cyclooxygenase-2 (COX-2), and rebuilding antioxidant/oxidation balance by reducing reactive oxygen species (ROS) and increasing Glutathione (GSH) in colon.

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.

Recent Advances of D-α-tocopherol Polyethylene Glycol 1000 Succinate Based Stimuli-responsive Nanomedicine for Cancer Treatment.

D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) is a pharmaceutical excipient approved by Chinese NMPA and FDA of USA. It's widely applied as a multifunctional drug carrier for nanomedicine. The advantages of TPGS include P-glycoprotein (P-gp) inhibition, penetration promotion, apoptosis induction via mitochondrial-associated apoptotic pathways, multidrug resistant (MDR) reversion, metastasis inhibition and so on. TPGS-based drug delivery systems which are responding to external stimulus can combine the inhibitory functions of TPGS towards P-gp with the environmentally responsive controlled release property and thus exerts a synergistic anti-cancer effect, through increased intracellular drug concentration in tumors cells and well-controlled drug release behavior. In this review, TPGS-based nano-sized delivery systems responsive to different stimuli were summarized and discussed, including pH-responsive, redoxresponsive and multi-responsive systems in various formulations. The achievements, mechanisms and different characteristics of TPGS-based stimuli-responsive drug-delivery systems in tumor therapy were also outlined.

In vitro and in vivo growth inhibition of human cervical cancer cells via human papillomavirus E6/E7 mRNAs' cleavage by CRISPR/Cas13a system.

Sustained infection of high-risk human papillomavirus (HR-HPVs), especially HPV16 and HPV18, is a major cause of cervical cancer. E6 and E7 oncoproteins, encoded by the HPV genome, are critical for transformation and maintenance of malignant phenotypes of cervical cancer. Here, we used an emerging programmable clustered regularly interspaced short palindromic repeat (CRISPR)/Cas13a system to cleave HPV 16/18 E6/E7 messenger RNAs (mRNAs). The results showed that customized CRISPR/Cas13a system effectively and specifically knocked down HPV 16/18 E6/E7 mRNAs, inducing growth inhibition and apoptosis in HPV16-positive SiHa and HPV18-positive HeLa Cell lines, but not in HPV-negative C33A cell line. Simultaneously, we detected downregulation of E6/E7 oncoproteins and upregulation of tumor suppressor P53 and RB proteins. In addition, we used subcutaneous xenograft tumor growth assays to find that the weight and volume of tumors in the SiHa-16E6CR1 group knocked down by the CRISPR/Cas13a system were significantly lower than those in the SiHa-VECTOR group lacking crRNA. Our study demonstrated that targeting HPV E6/E7 mRNAs by the CRISPR/Cas13a system may be a candidate therapeutic strategy for HPV-related cervical cancer.

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.