α, ω-Cholesterol-functionalized low molecular weight polyethylene glycol as a novel modifier of cationic liposomes for gene delivery.

Here, three novel cholesterol (Ch)/low molecular weight polyethylene glycol (PEG) conjugates, termed α, ω-cholesterol-functionalized PEG (Ch2-PEGn), were successfully synthesized using three kinds of PEG with different average molecular weight (PEG600, PEG1000 and PEG2000). The purpose of the study was to investigate the potential application of novel cationic liposomes (Ch2-PEGn-CLs) containing Ch2-PEGn in gene delivery. The introduction of Ch2-PEGn affected both the particle size and zeta potential of cationic liposomes. Ch2-PEG2000 effectively compressed liposomal particles and Ch2-PEG2000-CLs were of the smallest size. Ch2-PEG1000 and Ch2-PEG2000 significantly decreased zeta potentials of Ch2-PEGn-CLs, while Ch2-PEG600 did not alter the zeta potential due to the short PEG chain. Moreover, the in vitro gene transfection efficiencies mediated by different Ch2-PEGn-CLs also differed, in which Ch2-PEG600-CLs achieved the strongest GFP expression than Ch2-PEG1000-CLs and Ch2-PEG2000-CLs in SKOV-3 cells. The gene delivery efficacy of Ch2-PEGn-CLs was further examined by addition of a targeting moiety (folate ligand) in both folate-receptor (FR) overexpressing SKOV-3 cells and A549 cells with low expression of FR. For Ch2-PEG1000-CLs and Ch2-PEG2000-CLs, higher molar ratios of folate ligand resulted in enhanced transfection efficacies, but Ch2-PEG600-CLs had no similar in contrast. Additionally, MTT assay proved the reduced cytotoxicities of cationic liposomes after modification by Ch2-PEGn. These findings provide important insights into the effects of Ch2-PEGn on cationic liposomes for delivering genes, which would be beneficial for the development of Ch2-PEGn-CLs-based gene delivery system.

Development of lipid-shell and polymer core nanoparticles with water-soluble salidroside for anti-cancer therapy.

Salidroside (Sal) is a potent antitumor drug with high water-solubility. The clinic application of Sal in cancer therapy has been significantly restricted by poor oral absorption and low tumor cell uptake. To solve this problem, lipid-shell and polymer-core nanoparticles (Sal-LPNPs) loaded with Sal were developed by a double emulsification method. The processing parameters including the polymer types, organic phase, PVA types and amount were systemically investigated. The obtained optimal Sal-LPNPs, composed of PLGA-PEG-PLGA triblock copolymers and lipids, had high entrapment efficiency (65%), submicron size (150 nm) and negatively charged surface (-23 mV). DSC analysis demonstrated the successful encapsulation of Sal into LPNPs. The core-shell structure of Sal-LPNPs was verified by TEM. Sal released slowly from the LPNPs without apparent burst release. MTT assay revealed that 4T1 and PANC-1 cancer cell lines were sensitive to Sal treatment. Sal-LPNPs had significantly higher antitumor activities than free Sal in 4T1 and PANC-1 cells. The data indicate that LPNPs are a promising Sal vehicle for anti-cancer therapy and worthy of further investigation.

Multifunctional Mitochondria-Targeting Nanosystems for Enhanced Anticancer Efficacy.

Mitochondria, a kind of subcellular organelle, play crucial roles in cancer cells as an energy source and as a generator of reactive substrates, which concern the generation, proliferation, drug resistance, and other functions of cancer. Therefore, precise delivery of anticancer agents to mitochondria can be a novel strategy for enhanced cancer treatment. Mitochondria have a four-layer structure with a high negative potential, which thereby prevents many molecules from reaching the mitochondria. Luckily, the advances in nanosystems have provided enormous hope to overcome this challenge. These nanosystems include liposomes, nanoparticles, and nanomicelles. Here, we summarize the very latest developments in mitochondria-targeting nanomedicines in cancer treatment as well as focus on designing multifunctional mitochondria-targeting nanosystems based on the latest nanotechnology.

Association Between Genetic Polymorphisms of Metabolic Enzymes and Azathioprine-Induced Myelosuppression in 1,419 Chinese Patients: A Retrospective Study.

The aim of this study was to investigate the correlation between genetic polymorphisms of azathioprine-metabolizing enzymes and adverse reactions of myelosuppression. To this end, a retrospective analysis was performed on 1,419 Chinese patients involving 40 different diseases and 3 genes: ITPA (94C>A), TPMT*3 (T>C), and NUDT15 (415C>T). Strict inclusion and exclusion criteria were established to collect the relative cases, and the correlation between azathioprine and myelosuppression was evaluated by adverse drug reaction criteria. The mutation rates of the three genes were 29.32, 3.73, and 21.92% and grades I to IV myelosuppression occurred in 54 (9.28%) of the 582 patients who took azathioprine. The highest proportion of myelosuppression was observed in 5 of the 6 (83.33%) patients carrying the NUDT15 (415C>T) TT genotype and 12 of the 102 (11.76%) patients carrying the NUDT15 (415C>T) CT genotype. Only the NUDT15 (415C>T) polymorphism was found to be associated with the adverse effects of azathioprine-induced myelosuppression (odds ratio [OR], 51.818; 95% CI, 5.280-508.556; p = 0.001), which suggested that the NUDT15 (415C>T) polymorphism could be an influencing factor of azathioprine-induced myelosuppression in the Chinese population. Epistatic interactions between ITPA (94C>A) and NUDT15 (415C>T) affect the occurrence of myelosuppression. Thus, it is recommended that the genotype of NUDT15 (415C>T) and ITPA (94C>A) be checked before administration, and azathioprine should be avoided in patients carrying a homozygous NUDT15 (415C>T) mutation. This study is the first to investigate the association between genetic polymorphisms of these three azathioprine-metabolizing enzymes and myelosuppression in a large number of cases with a diverse range of diseases.

The approved gene therapy drugs worldwide: from 1998 to 2019.

With the improvement of gene vectors, the rise of chimeric antigen receptor T cell immunotherapy and breakthroughs in the genome editing technology, gene therapy had once again returned to the central stage of disease treatment. It had brought new choices to clinical therapy of diseases such as tumors and genetic diseases, and had changed the status quo of treatment for monogenic disorders and diffuse large B-cell lymphoma. Until August 2019, 22 gene medicines had been approved by the drug regulatory agencies from various countries, but there were few relevant reviews of combing these drugs systematically. Consequently, this review summarizes the gene therapy drugs approved worldwide from 1998 to 2019 in details, including names, indications, dates of approval, companies, vectors, the applied technologies and mechanisms of gene therapy drugs, etc. Furthermore, the gene therapy drugs were classified and addressed in accordance with the employed vectors. Gene therapy had gradually been accepted by the government and the public since 1980s, and have become a new and important alternative to existing treatments for human diseases in the past few years. Therefore, gene therapy drugs, with safe vectors and advanced biotechnologies, would play a greater role in the prevention and treatment of human diseases in future.

Novel zwitterionic vectors: Multi-functional delivery systems for therapeutic genes and drugs.

Zwitterions consist of equal molar cationic and anionic moieties and thus exhibit overall electroneutrality. Zwitterionic materials include phosphorylcholine, sulfobetaine, carboxybetaine, zwitterionic amino acids/peptides, and other mix-charged zwitterions that could form dense and stable hydration shells through the strong ion-dipole interaction among water molecules and zwitterions. As a result of their remarkable hydration capability and low interfacial energy, zwitterionic materials have become ideal choices for designing therapeutic vectors to prevent undesired biosorption especially nonspecific biomacromolecules during circulation, which was termed antifouling capability. And along with their great biocompatibility, low cytotoxicity, negligible immunogenicity, systematic stability and long circulation time, zwitterionic materials have been widely utilized for the delivery of drugs and therapeutic genes. In this review, we first summarized the possible antifouling mechanism of zwitterions briefly, and separately introduced the features and advantages of each type of zwitterionic materials. Then we highlighted their applications in stimuli-responsive "intelligent" drug delivery systems as well as tumor-targeting carriers and stressed the multifunctional role they played in therapeutic gene delivery.

Understanding the sheet size-antibacterial activity relationship of graphene oxide and the nano-bio interaction-based physical mechanisms.

The antibiotics-independent antimicrobial activity of graphene oxide (GO) is of great importance since antibiotic therapy is facing great challenges from drug resistance. However, the relations of GO size with its antimicrobial activity and how the size regulates the antibacterial mechanisms are still unknown. Herein, we fabricated four GO suspensions with different sizes and demonstrated the parabolic relationship between GO size and its antibacterial activity against the Gram-positive cariogenic bacterium Streptococcus mutans. More interestingly, we found out how GO size regulated the nano-bio interaction-based physical antibacterial mechanisms. Increasing the size reduced the cutting effect but enhanced the cell entrapment effect, and vice versa. In conclusion, GO size affects its edge density and lateral dimension, further regulates its physical antibacterial mechanisms in different orientations and ultimately determines its activity. These findings provide a deep understanding of GO antibacterial property and may guide the design and development of GO for clinical use.

Current RNA-based Therapeutics in Clinical Trials.

Long-term research on various types of RNAs has led to further understanding of diverse mechanisms, which eventually resulted in the rapid development of RNA-based therapeutics as powerful tools in clinical disease treatment. Some of the developing RNA drugs obey the antisense mechanisms including antisense oligonucleotides, small interfering RNAs, microRNAs, small activating RNAs, and ribozymes. These types of RNAs could be utilized to inhibit/activate gene expression or change splicing to provide functional proteins. In the meantime, some others based on different mechanisms like modified messenger RNAs could replace the dysfunctional endogenous genes to manage some genetic diseases, and aptamers with special three-dimensional structures could bind to specific targets in a high-affinity manner. In addition, the recent most popular CRISPR-Cas technology, consisting of a crucial single guide RNA, could edit DNA directly to generate therapeutic effects. The desired results from recent clinical trials indicated the great potential of RNA-based drugs in the treatment of various diseases, but further studies on improving delivery materials and RNA modifications are required for the novel RNA-based drugs to translate to the clinic. This review focused on the advances and clinical studies of current RNA-based therapeutics, analyzed their challenges and prospects.

Progress in the treatment of infantile hemangioma.

Infantile hemangioma (IH) is a common benign tumor, which mostly resolves spontaneously; however, children with high-risk IH need treatment. Currently, the recognized first-line treatment regimen for IH is oral propranolol, but research on the pathogenesis of IH has led to the identification of new therapeutic targets, which have shown good curative effects, providing more options for disease treatment. This article summarizes the applications of different medications, dosages, and routes of administration for the treatment of IH. In addition to drug therapy, this article also reviews current therapeutic options for IH such as laser therapy, surgical treatment, and observation. To provide the best treatment, therapeutic regimens for IH should be selected based on the child's age, the size and location of the lesion, the presence of complications, the implementation conditions, and the potential outcomes of the treatment.

Current Advances in Small Activating RNAs for Gene Therapy: Principles, Applications and Challenges.

Small activating RNAs (saRNAs) are small double-stranded RNAs that could mediate the target-specific gene expression by targeting selected sequences in gene promoters at both the transcriptional and epigenetic levels. This phenomenon of gene manipulation is known as RNA activation (RNAa), which opens up a new pathway for RNA-based gene therapeutics in contrast to RNA interference. Although the exact molecular mechanism of RNAa mediated by saRNAs still remains foggy, some studies have provided the possible ones to explain it. Furthermore, mounting evidence exhibit that saRNAs not only provide a new approach to study gene function and manipulate transcriptional activity, but also promise a great potential for clinical therapy against various diseases, especially cancer. Cancer-associated genes could be up-regulated by saRNAs to modulate cell cycle and proliferation, induce cell senescence and apoptosis, inhibit cancer cell invasion and migration, and reverse chemotherapy resistance. Herein, we summarize the known mechanisms of saRNAs on up-regulating specific gene expression and focus on the potential applications of saRNAs in gene therapy. In addition, some concerns about mechanisms and challenges for delivery of saRNAs are involved in this review. The precise mechanisms of saRNAs need to be further illustrated and some novel delivery systems for saRNAs are expected to be developed for clinical applications.

In Vivo Ovarian Cancer Gene Therapy Using CRISPR-Cas9.

Clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9) genome editing technology holds great promise for the field of human gene therapy. However, a lack of safe and effective delivery systems restricts its biomedical application. Here, a folate receptor-targeted liposome (F-LP) was used to deliver CRISPR plasmid DNA co-expressing Cas9 and single-guide RNA targeting the ovarian cancer-related DNA methyltransferase 1 (DNMT1) gene (gDNMT1). F-LP efficiently bound the gDNMT1 plasmid and formed a stable complex (F-LP/gDNMT1) that was safe for injection. F-LP/gDNMT1 effectively mutated endogenous DNMT1 in vitro, and then expressed the Cas9 endonuclease and downregulated DNMT1 in vivo. The tumor growth of both paclitaxel-sensitive and -resistant ovarian cancers were inhibited by F-LP/gDNMT1, which shows fewer adverse effects than paclitaxel injection. Therefore, CRISPR-Cas9-targeted DNMT1 manipulation may be a potential therapeutic regimen for ovarian cancer, and lipid-mediated delivery systems represent promising delivery vectors of CRISPR-Cas9 technology for precise genome editing therapeutics.

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

Recent advances of biomaterials in biotherapy.

Biotherapy mainly refers to the intervention and the treatment of major diseases with biotechnologies or bio-drugs, which include gene therapy, immunotherapy (vaccines and antibodies), bone marrow transplantation and stem-cell therapy. In recent years, numerous biomaterials have emerged and were utilized in the field of biotherapy due to their biocompatibility and biodegradability. Generally, biomaterials can be classified into natural or synthetic polymers according to their source, both of which have attracted much attention. Notably, biomaterials-based non-viral gene delivery vectors in gene therapy are undergoing rapid development with the emergence of surface-modified or functionalized materials. In immunotherapy, biomaterials appear to be attractive means for enhancing the delivery efficacy and the potency of vaccines. Additionally, hydrogels and scaffolds are ideal candidates in stem-cell therapy and tissue engineering. In this review, we present an introduction of biomaterials used in above biotherapy, including gene therapy, immunotherapy, stem-cell therapy and tissue engineering. We also highlighted the biomaterials which have already entered the clinical evaluation.

Recent Advances of Poly(ether-ether) and Poly(ether-ester) Block Copolymers in Biomedical Applications.

BACKGROUND: Poly(ether-ether) and poly(ether-ester) block copolymers have been widely applied in biomedical fields over two decades due to their good safety and biocompatibility. Poly(ethylene glycol), poly(ethylene glycol)-poly(propylene glycol) and poly(lactic-co-glycolic acid) have been approved as excipients by Food and Drug Administration. Because of the broad perspective in biomedical fields, many novel poly(etherether) and poly(ether-ester) block copolymers have been developed for drug delivery, gene therapy and tissue engineering in recent years. This review focuses on active targeting theranostic systems, gene delivery systems and tissue engineering based on poly(ether-ether) and poly(ether-ester) block copolymers. METHODS: We perform a structured search of bibliographic databases for peer-reviewed scientific reports using a focused review question and inclusion/exclusion criteria. The literatures related to the topics of this review are cataloged according to the developed copolymers or their applications such as active targeting theranostic systems, gene delivery systems and tissue engineering. Some important advances and new trends are summarized in this review. RESULTS: Some commercial poly(ether-ether) copolymers have been used as excipients for drug research and development. Amphiphilic and biodegradable poly(ether-ester) diblock copolymers are capable of formulating biomedical nanoparticulate theranostic systems, and targeting moiety-functionalized poly(ether-ester) diblock copolymers will be further developed and applied in biomedical nanotechnology fields in the near future. Meanwhile, triblock or multiblock poly(ether-ether) and poly(ether-ester) copolymers with environmentsensitive properties are suitable for gene delivery and tissue engineering. Poly(ether-ether) and poly(ether-ester) copolymers are being extensively applied in active targeting theranostic systems, gene delivery systems and tissue engineering. CONCLUSIONS: Biodegradable, environment-sensitive and targeting moiety-functionalized block copolymers, which are being applied in active targeting theranostic systems, gene delivery systems and tissue engineering, are promising candidates for treatment of various diseases.

A folate receptor-targeted lipoplex delivering interleukin-15 gene for colon cancer immunotherapy.

Interleukin-15 has been implicated as a promising cytokine for cancer immunotherapy, while folate receptor α (FRα) has been shown to be a potentially useful target for colon cancer therapy. Herein, we developed F-PLP/pIL15, a FRα-targeted lipoplex loading recombinant interleukin-15 plasmid (pIL15) and studied its antitumor effects in vivo using a CT26 colon cancer mouse model. Compared with control (normal saline) treatment, F-PLP/pIL15 significantly suppressed tumor growth in regard to tumor weight (P < 0.001) and reduced tumor nodule formation (P < 0.001). Moreover, when compared to other lipoplex-treated mice, F-PLP/pIL15-treated mice showed higher levels of IL15 secreted in the serum (P < 0.001) and ascites (P < 0.01). These results suggested that the targeted delivery of IL15 gene might be associated with its in vivo antitumor effects, which include inducing tumor cell apoptosis, inhibiting tumor proliferation and promoting the activation of immune cells such as T cells and natural killer cells. Furthermore, hematoxylin and eosin staining of vital organs following F-PLP/pIL15 treatment showed no detectable toxicity, thus indicating that intraperitoneal administration may be a viable route of delivery. Overall, these results suggest that F-PLP/pIL15 may serve as a potential targeting preparation for colon cancer therapy.

Ovarian cancer treatment with a tumor-targeting and gene expression-controllable lipoplex.

Overexpression of folate receptor alpha (FRα) and high telomerase activity are considered to be the characteristics of ovarian cancers. In this study, we developed FRα-targeted lipoplexes loaded with an hTERT promoter-regulated plasmid that encodes a matrix protein (MP) of the vesicular stomatitis virus, F-LP/pMP(2.5), for application in ovarian cancer treatment. We first characterized the pharmaceutical properties of F-LP/pMP(2.5). The efficient expression of the MP-driven hTERT promoter in SKOV-3 cells was determined after an in-vitro transfection assay, which was significantly increased compared with a non-modified LP/pMP(2.5) group. F-LP/pMP(2.5) treatment significantly inhibited the growth of tumors and extended the survival of mice in a SKOV-3 tumor model compared with other groups. Such an anti-tumor effect was due to the increased expression of MP in tumor tissue, which led to the induction of tumor cell apoptosis, inhibition of tumor cell proliferation and suppression of tumor angiogenesis. Furthermore, a preliminary safety evaluation demonstrated a good safety profile of F-LP/pMP(2.5) as a gene therapy agent. Therefore, FRα-targeted lipoplexes with therapeutic gene expression regulated by an hTERT promoter might be a promising gene therapy agent and a potential translational candidate for the clinical treatment of ovarian cancer.