Multistage targeting and dual inhibiting strategies based on bioengineered tumor matrix microenvironment-mediated protein nanocages for enhancing cancer biotherapy.

Regulation of the apoptotic pathway plays a critical role in inducing tumor cell death and circumventing drug resistance. Survivin protein is the strongest inhibitor of apoptosis found so far. It is highly expressed in several cancers and is a promising target for cancer therapy. However, clinical applications are limited by incomplete inhibition of survivin expression. Here, we present a novel strategy that extended the release of YM155 (an effective survivin inhibitor that works by inhibiting the activity of survivin promoter) and TATm-survivin (T34A) (TmSm) protein (survivin protein mutant with penetrating peptide, a potential anticancer protein therapeutic) via tumor matrix microenvironment-mediated ferritin heavy chain nanocages (FTH1 NCs), enabling significant inhibition of survivin activity at both transcript and protein levels. FTS (FTH1-matrix metalloproteinase-2-TmSm)/YM155 NC synthesis was easily scaled up, and these NCs could sequentially release TmSm protein through matrix metalloproteinase-2 and promote YM155 to enter the nucleus via transferrin receptor 1 (TfR1) binding, which increased the cytotoxicity and apoptosis of Capan-2 and A549 cells compared to that with individual drugs. Moreover, FTS/YM155 NCs enhanced drug accumulation at tumor sites and had a higher tumor inhibition rate (88.86%) than the compounds alone in A549 tumor-bearing mice. In addition, FTS/YM155 NCs exerted significant survivin downregulation (4.43-fold) and caspase-3 upregulation (4.31-fold) and showed better therapeutic outcomes without inducing organ injury, which highlights their promising future clinical application in precision therapy. This tumor microenvironment-responsive platform could be harnessed to develop an effective therapy via multilevel inhibition of cancer targets.

Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs.

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

Designing and constructing a phage display synthesized single domain antibodies library based on camel VHHs frame for screening and identifying humanized TNF-α-specific nanobody.

Tumor necrosis factor (TNF-α) is an important clinically tested cytokine that could induce autoimmune diseases and inflammation. Therefore, the anti-TNF-α therapy strategy was developed and used therapeutically in various diseases, especially in the cytokine storm associated chimeric antigen receptor (CAR) T-cell therapy and antiviral therapy. Compare with other anti-TNF-α inhibitors, anti-TNF-α Nb (nanobody) has many unique advantages. Herein, we reported a novel humanized scaffold for library construction, which could be soluble and expressed in Escherichia coli (E.coli), and the efficiency capacity could reach as high as 2.01 × 109. Meanwhile, an anti-TNF-α Nb was selected for further study after 4 rounds of screening, NT-3, as the optimal Nb could effectively inhibit TNF-mediated cytotoxicity. The IC50 of NT-3 was determined as 0.804 µM, and its apoptosis inhibition rate was 62.47 % in L929 cells. Furthermore, the molecular docking results showed that complementarity-determining regions (CDRs) of NT-3 could connect to TNF for blocking function through strong hydrogen bonds and salt bridges. In general, our study not only provided a good Nb screening platform in vitro without animal immunization, but also generated a series of novel humanized anti-TNF-α Nb candidates with potential applications.

PEGylated-PLGA Nanoparticles Coated with pH-Responsive Tannic Acid-Fe(III) Complexes for Reduced Premature Doxorubicin Release and Enhanced Targeting in Breast Cancer.

Biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been widely used as delivery vehicles for chemotherapy drugs. However, premature drug release in PLGA NPs can damage healthy tissue and cause serious adverse effects during systemic administration. Here, we report a tannic acid-Fe(III) (FeIII-TA) complex-modified PLGA nanoparticle platform (DOX-TPLGA NPs) for the tumor-targeted delivery of doxorubicin (DOX). A PEGylated-PLGA inner core and FeIII-TA complex outer shell were simultaneously introduced to reduce premature drug release in blood circulation and increase pH-triggered drug release in tumor tissue. Compared to the unmodified NPs, the initial burst rate of DOX-TPLGA NPs was significantly reduced by nearly 2-fold at pH 7.4. Moreover, the cumulative drug release rate at pH 5.0 was 40% greater than that at pH 7.4 due to the pH-response of the FeIII-TA complex. Cellular studies revealed that the TPLGA NPs had enhanced drug uptake and superior cytotoxicity of breast cancer cells in comparison to free DOX. Additionally, the DOX-TPLGA NPs efficiently accumulated in the tumor site of 4T1-bearing nude mice due to the enhanced permeability and retention (EPR) effect and reached a tumor inhibition rate of 85.53 ± 8.77% (1.31-fold versus DOX-PLGA NPs and 3.12-fold versus free DOX). Consequently, the novel TPLGA NPs represent a promising delivery platform to enhance the safety and efficacy of chemotherapy drugs.

The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance.

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

A novel liposome-polymer hybrid nanoparticles delivering a multi-epitope self-replication DNA vaccine and its preliminary immune evaluation in experimental animals.

DNA vaccine is an attractive immune platform for the prevention and treatment of infectious diseases, but existing disadvantages limit its use in preclinical and clinical assays, such as weak immunogenicity and short half-life. Here, we reported a novel liposome-polymer hybrid nanoparticles (pSFV-MEG/LNPs) consisting of a biodegradable core (mPEG-PLGA) and a hydrophilic shell (lecithin/PEG-DSPE-Mal 2000) for delivering a multi-epitope self-replication DNA vaccine (pSFV-MEG). The pSFV-MEG/LNPs with optimal particle size (161.61 ±â€¯15.63 nm) and high encapsulation efficiency (87.60 ±â€¯8.73%) induced a strong humoral (3.22-fold) and cellular immune responses (1.60-fold) compared to PBS. Besides, the humoral and cellular immune responses of pSFV-MEG/LNPs were 1.58- and 1.05-fold than that of pSFV-MEG. All results confirmed that LNPs was a very promising tool to enhance the humoral and cellular immune responses of pSFV-MEG. In addition, the rational design and delivery platform can be used for the development of DNA vaccines for other infectious diseases.

Multiple targeting strategies achieve novel protein drug delivery into proapoptosis lung cancer cells by precisely inhibiting survivin.

Therapeutic recombinant proteins have numerous advantages and benefits over chemical drugs, particularly high specificity and good biocompatibility. However, the therapeutic potential and clinical application of current anticancer protein drugs are limited as most biomarkers are located within cells, and multiple physiological barriers exist between the point of administration and the intracellular biomarker. Herein, we report a novel strategy to accurately deliver a cell-permeable dominant-negative TATm-Survivin (TmSm) protein (T34A) to intracellular survivin in cancer cells by overcoming multiple barriers in vivo. A poly(d,l-lactide-co-glycolide) (PLGA) inner core, a polyethylene glycol (PEG) modification, and a TATm peptide were simultaneously introduced to mediate tumor tissue targeting and response to pH-triggered TmSm release. Compared to free TmSm, the PEGylated-PLGA nanoparticle platform achieved a significantly higher cellular uptake efficiency (1.79-fold for A549 and 1.77-fold for Capan-2), effectively decreased IC50 (1.22-fold for A549 and 1.17-fold for Capan-2), and largely elevated apoptosis in different cancer cells (1.17-fold for A549 and 1.15-fold for Capan-2). Besides, this newly developed nanoplatform showed increased protein drug accumulation in the tumor site in A549-bearing nude mice and reached a tumor inhibition rate of 55.81% (1.35-fold versus free TmSm) by reducing the expression of intracellular survivin. All these results confirmed that our newly developed delivery strategy is a very promising tool, which helps protein drugs to cross multiple barriers in vivo and achieves precise targeting to intracellular biomarkers. This strategy could also be applied to other types of protein drugs to further improve their clinical anticancer therapeutic efficacy.

Elucidating Bottlenecks to the Efficient Preparation of AB5-Hexamer Mucosal Adjuvant Protein LTm by Genetic Engineering.

Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli, we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein.

Elucidating respective functions of two domains BIR and C-helix of human IAP survivin for precise targeted regulating mitotic cycle, apoptosis and autophagy of cancer cells.

Survivin was the smallest member of the IAP family, which was over expressed in many different cancers, and considered to be a promising hot target for cancer therapy, and our previous study demonstrated that multiple dominant negative mutants from full-length survivin could have many complex effects on cancer cells, such as cell cycle, apoptosis, and autophagy. But it was not yet known what role the two main domains played in those functions, which would be very important for the design of targeted anticancer drugs and for the interpretation of their molecular mechanisms. In this study, based on preparation the two parts (BIR domain and CC domain) of survivin by genetic engineering and cell characterization assay, we discovered that BIR (T34A)-domain peptide could inhibit Bcap-37 cells growth in a dose- and time-dependent manner, increase the proportion of G2/M phase, and induce caspase-dependent apoptosis via the mitochondrial pathway. While CC (T117A)-domain peptide increased the proportion of S-phase cells and increased the level of the autophagy marker protein LC3B significantly. These further experiments confirmed that TAT-BIR (T34A) peptide could be used to inhibit cell proliferation, promote apoptosis, and block mitosis, and TAT-CC (T117A) peptide showed mainly to promote autophagy, process of DNA replication, and mitosis to breast cancer cells. This research will lay the foundation for interpreting the multifunction mechanism of survivin in cell fates, further make senses in developing the anticancer drugs targeting it precisely and efficiently.