A pH-responsive cetuximab-conjugated DMAKO-20 nano-delivery system for overcoming K-ras mutations and drug resistance in colorectal carcinoma.

Cetuximab (Cet) and oxaliplatin (OXA) are used as first-line drugs for patients with colorectal carcinoma (CRC). In fact, the heterogeneity of CRC, mainly caused by K-ras mutations and drug resistance, undermines the effectiveness of drugs. Recently, a hydrophobic prodrug, (1E,4E)-6-((S)-1-(isopentyloxy)-4-methylpent-3-en-1-yl)-5,8-dimethoxynaphthalene-1,4­dione dioxime (DMAKO-20), has been shown to undergo tumor-specific CYP1B1-catalyzed bioactivation. This process results in the production of nitric oxide and active naphthoquinone mono-oximes, which exhibit specific antitumor activity against drug-resistant CRC. In this study, a Cet-conjugated bioresponsive DMAKO-20/PCL-PEOz-targeted nanocodelivery system (DMAKO@PCL-PEOz-Cet) was constructed to address the issue of DMAKO-20 dissolution and achieve multitargeted delivery of the cargoes to different subtypes of CRC cells to overcome K-ras mutations and drug resistance in CRC. The experimental results demonstrated that DMAKO@PCL-PEOz-Cet efficiently delivered DMAKO-20 to both K-ras mutant and wild-type CRC cells by targeting the epidermal growth factor receptor (EGFR). It exhibited a higher anticancer effect than OXA in K-ras mutant cells and drug-resistant cells. Additionally, it was observed that DMAKO@PCL-PEOz-Cet reduced the expression of glutathione peroxidase 4 (GPX4) in CRC cells and significantly inhibited the growth of heterogeneous HCT-116 subcutaneous tumors and patient-derived tumor xenografts (PDX) model tumors. This work provides a new strategy for the development of safe and effective approaches for treating CRC. STATEMENT OF SIGNIFICANCE: (1) Significance: This work reports a new approach for the treatment of colorectal carcinoma (CRC) using the bioresponsible Cet-conjugated PCL-PEOz/DMAKO-20 nanodelivery system (DMAKO@PCL-PEOz-Cet) prepared with Cet and PCL-PEOz for the targeted transfer of DMAKO-20, which is an anticancer multitarget drug that can even prevent drug resistance, to wild-type and K-ras mutant CRC cells. DMAKO@PCL-PEOz-Cet, in the form of nanocrystal micelles, maintained stability in peripheral blood and efficiently transported DMAKO-20 to various subtypes of colorectal carcinoma cells, overcoming the challenges posed by K-ras mutations and drug resistance. The system's secure and effective delivery capabilities have also been confirmed in organoid and PDX models. (2) This is the first report demonstrating that this approach simultaneously overcomes the K-ras mutation and drug resistance of CRC.

MDSC-targeted liposomal all-trans retinoic acid suppresses mMdscs and improves immunotherapy in HBV infection.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are evolving as a prominent determinant in cancer occurrence and development and are functionally found to suppress T cells in cancer. Not much research is done regarding its involvement in viral infections. This research was designed to investigate the role of MDSCs in hepatitis B virus (HBV) infection and how targeting these cells with our novel all-trans retinoic acid encapsulated liposomal formulation could improve immunotherapy in C57BL/6 mice. METHODS: Ten micrograms (10 µg) of plasmid adeno-associated virus (pAAV/HBV 1.2, genotype A) was injected hydrodynamically via the tail vein of C57BL/6 mice. An all-trans retinoic acid encapsulated liposomal formulation (L-ATRA) with sustained release properties was used in combination with tenofovir disoproxil fumarate (TDF), a nucleotide analog reverse transcriptase inhibitor (nRTI) to treat the HBV infection. The L-ATRA formulation was given at a dose of 5 mg/kg intravenously (IV) twice a week. The TDF was given orally at 30 mg/kg daily. RESULTS: Our results revealed that L-ATRA suppresses MDSCs in HBV infected mice and enhanced T-cell proliferation in vitro. In vivo studies showed higher and improved immunotherapeutic effect in mice that received L-ATRA and TDF concurrently in comparison with the groups that received monotherapy. Lower HBV DNA copies, lower concentrations of HBsAg and HBeAg, lower levels of ALT and AST and less liver damage were seen in the mice that received the combination therapy of L-ATRA + TDF. CONCLUSIONS: In effect, targeting MDSCs with the combination of L-ATRA and TDF effectively reduced mMDSC and improved immunotherapy in the HBV infected mice. Targeting MDSCs could provide a breakthrough in the fight against hepatitis B virus infection.

Precision Therapy of Recurrent Breast Cancer through Targeting Different Malignant Tumor Cells with a HER2/CD44-Targeted Hydrogel Nanobot.

Heterogeneity and drug resistance of tumor cells are the leading causes of incurability and poor survival for patients with recurrent breast cancer. In order to accurately deliver the biological anticancer drugs to different subtypes of malignant tumor cells for omnidirectional targeted treatment of recurrent breast cancer, a distinct design is demonstrated by embedding liposome-based nanocomplexes containing pro-apoptotic peptide and survivin siRNA drugs (LPR) into Herceptin/hyaluronic acid cross-linked nanohydrogels (Herceptin-HA) to fabricate a HER2/CD44-targeted hydrogel nanobot (named as ALPR). ALPR delivered cargoes to the cells overexpressing CD44 and HER2, followed by Herceptin-HA biodegradation, subsequently, the exposed lipid component containing DOPE fused with the endosomal membrane and released peptide and siRNA into the cytoplasm. These experiments indicated that ALPR can specifically deliver Herceptin, peptide, and siRNA drugs to HER2-positive SKBR-3, triple-negative MDA-MB-231, and HER2-negative drug-resistant MCF-7 human breast cancer cells. ALPR completely inhibited the growth of heterogeneous breast tumors via multichannel synergistic effects: disrupting mitochondria, downregulating the survivin gene, and blocking HER2 receptors on the surface of HER2-positive cells. The present design overcomes the chemical drug resistance and opens a feasible route for the combinative treatment of recurrent breast cancer, even other solid tumors, utilizing different kinds of biological drugs.

Targeted Therapy and Immunotherapy for Heterogeneous Breast Cancer.

Breast cancer (BC) is the most common malignancy in women worldwide, and it is a molecularly diverse disease. Heterogeneity can be observed in a wide range of cell types with varying morphologies and behaviors. Molecular classifications are broadly used in clinical diagnosis, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and breast cancer gene (BRCA) mutations, as indicators of tumor heterogeneity. Treatment strategies differ according to the molecular subtype. Besides the traditional treatments, such as hormone (endocrine) therapy, radiotherapy, and chemotherapy, innovative approaches have accelerated BC treatments, which contain targeted therapies and immunotherapy. Among them, monoclonal antibodies, small-molecule inhibitors and antibody-drug conjugates, and targeted delivery systems are promising armamentarium for breast cancer, while checkpoint inhibitors, CAR T cell therapy, cancer vaccines, and tumor-microenvironment-targeted therapy provide a more comprehensive understanding of breast cancer and could assist in developing new therapeutic strategies.

Assembly strategy of liposome and polymer systems for siRNA delivery.

In recent years, gene therapy has made tremendous progress in the development of disease treatment. Among them, siRNA offers specificity of gene silencing, ease of synthesis, and short development period, and has been intensively studied worldwide. However, siRNA as the hydrophilic polyanion is easily degraded in vivo and poorly taken up into cells and so, the benefits of its powerful gene silencing ability will not be realized until better carriers are developed that are capable of protecting siRNA and delivering it intact to the cytoplasm of the target cells. Cationic liposomes (CL) and cationic polymers (CP) are the main non-viral siRNA vectors, there have been a lot of reports on the use of these two carriers to deliver siRNA. Whereas, as far as we know, there have been few review articles that provide an in-depth summary of the siRNA loading principle and internal structures of the siRNA delivery system. We summarize the formation principle and assembly structure of the cationic liposome-siRNA and polymer-siRNA complexes, and point out their advantages and characteristics and also show how to perfect their assembly and improve their clinical application in the future. It supports some useful suggestions for siRNA therapy, specifically, safe and efficient delivery.

Skin microbiota analysis-inspired development of novel anti-infectives.

BACKGROUND: The alarming spread of antimicrobial resistance requires the development of novel anti-infective drugs. Despite the recent research focus on the human microbiome and its likely value to understand and exploit inter-bacterial inhibitory phenomena as a source for antimicrobial strategies, the human microbiota has barely been investigated for the purpose of drug development. RESULTS: We performed a large screen analyzing over 3000 human skin isolates to evaluate bacterial competition within the human skin microbiota as a basis for the development of anti-infective therapeutics. We discovered a Staphylococcus hominis strain with strong and broad activity against Gram-positive pathogens that was mediated by the bacteriocin micrococcin P1 (MP1). In "probiotic" approaches, this strain led to reduced Staphylococcus aureus infection and accelerated closure of S. aureus-infected wounds. Furthermore, we used a nanoparticle strategy to overcome the physico-chemical limitations often encountered with natural substances such as MP1 and demonstrate a significant reduction of S. aureus infection by MP1-loaded nanoparticles. CONCLUSIONS: Our study gives examples of how analysis of bacterial interactions in the human microbiota can be explored for the development of novel, effective anti-infective strategies. Video Abstract.

Herceptin-conjugated paclitaxel loaded PCL-PEG worm-like nanocrystal micelles for the combinatorial treatment of HER2-positive breast cancer.

We have constructed Herceptin-conjugated, paclitaxel (PTX) loaded, PCL-PEG worm-like nanocrystal micelles (PTX@PCL-PEG-Herceptin) for the combinatorial therapy of HER2-positive breast cancer that exploit the specific targeting of Herceptin to HER2-positive breast cancer cells. Firstly, amphiphilic PCL2000-MPEG2000 and PCL5000-PEG2000-CHO were selected as the optimized matrix to wrap PTX that self-assembled into worm-like micelles with internal nanocrystal structures (PTX@PCL-PEG). Then the aldehydes of PCL5000-PEG2000-CHO exposed on the outside surface of PTX@PCL-PEG were utilized to react with the primary amines of Herceptin and formed stable, carbon-nitrogen single linkers (-C-N-) between the antibodies and nanoparticles. This study shows PTX@PCL-PEG-Herceptin remained relatively stable in the circulation and in the tumor microenvironment, and rapidly targeted and entered into the HER2-overexpressing tumor cells while sparing normal tissues from the toxic effects. PTX@PCL-PEG-Herceptin shrank the tumors and prolonged survival time in a SKBR-3-tumor-xenograft, nude mice model more effectively than TAXOL®, PTX@PCL-PEG, Herceptin+TAXOL® and Herceptin+PTX@PCL-PEG. Mechanistic studies showed that PTX@PCL-PEG-Herceptin entered into the HER2-positive tumor cells through the caveolin-mediated pathway. The conjugated Herceptin greatly enhanced the binding ability of the nanoparticle to the targeted SKBR-3 cells. This novel strategy provides a rational and simple antibody-conjugated-nanoparticle platform for the clinical application of combinatorial anticancer treatment.

A general platform for efficient extracellular expression and purification of Fab from Escherichia coli.

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.

Smart nanoparticles assembled by endogenous molecules for siRNA delivery and cancer therapy via CD44 and EGFR dual-targeting.

We developed an anticancer siRNA delivery system (named HLPR) through modular assembly of endogenous molecules. The structure of HLPR was a tightly condensed siRNA-peptide inner core in turn surrounded by the disordered lipid layer and thin HA coating from which the EGFR-targeted amino acid sequences of YHWYGYTPQNVI partially protrude outside of cell surfaces. Both HA and YHWYGYTPQNVI anchored on HLPR were responsible for targeting CD44 and EGFR overexpressed on the tumor cell surfaces, respectively. HLPR was relatively stable in the blood circulation and reached the tumor tissue in vivo through passive and active targeting. Then HLPR entered tumor cells mainly through EGFR-mediated pathway followed by the separation of HA from the remaining parts of nanocomplexes. The HA-uncoated complexes escaped the endosome through the membrane fusion function of DOPE and released cargoes (siRNA and peptide/siRNA) in the cytoplasm. HLPR significantly inhibited the growth of implanted subcutaneous liver tumors without toxicity.

Hepatic Carcinoma Selective Nucleic Acid Nanovector Assembled by Endogenous Molecules Based on Modular Strategy.

We rationally formulated a nucleic acid nanovector platform utilizing endogenous molecules in the following steps: nucleic acids are initially packed by a multifunctional peptide and a cationic liposome to form positively charged ternary complexes through electrostatic interaction; then the ternary complexes were coated with hyaluronic acid (HA) to form negatively charged quaternary nanocomplexes (Q-complexes). Among the components of Q-complexes, the multifunctional peptide was composed of a poly-16-arginine (R16) and a hepatic tumor-targeted cell penetrating peptide (KRPTMRFRYTWNPMK); the cationic lipid component included DOTAP and fusogenic lipid DOPE; the HA component shielded the cationic ternary complexes and actively targeted the CD44 overexpressed on the surface of tumor cells. Q-complexes have showed a relatively high stability in the medium, and HA component partially separated from the nanocomplexes after the Q-complexes bound to the cancer cells. The Q-complexes showed significantly enhanced nucleic acid delivery activity than the corresponding quaternary complexes containing R16 and nonvisible cytotoxicity in SCMM-7721 cells. In vivo, a selected Q-complex HLP1R specifically targeted and entered tumor cells without affecting normal tissues. Furthermore, HLP1R wrapped survivin siRNA efficiently and silenced the targeting gene in the liver orthotropic transplantation tumor models and showed nontoxic in vivo. This study reveals that Q-complexes are reasonable and feasible gene therapeutic carriers.

Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design.

A safe and efficient quaternary gene delivery system (named Q-complexes) was constructed based on self-assembly of molecules through noncovalent bonds. This system was formulated through the cooperation and competing interactions of cationic liposomes, multifunctional peptides, and DNA, followed by coating hyaluronic acid on the surface of the ternary complexes. The multifunctional peptide was composed of two functional domains: penetrating hepatic tumor-targeted cell moiety (KRPTMRFRYTWNPMK) and a wrapping gene sequence (polyarginine 16). The effect of spacer insertion between the two domains of multifunctional peptide on the intracellular transfection of Q-complexes was further studied. Experimental results showed that the formulations assembled with various peptides in the spacer elements possessed different intercellular pathways and transfection efficiencies. The Q-complexes containing peptide in the absence of spacer element (Pa) showed the highest gene expression among all samples. The Q-complexes containing peptides with a noncleavable spacer GA (Pc) had no ability of intracellular nucleic acid delivery, whereas those with a cleavable spacer RVRR (Pd) showed moderate transfection activity. These results demonstrated that the different spacers inserted in the multifunctional peptide played an important role in in vitro DNA transfection efficiency. Atomic force microscopy images showed that the morphologies of ternary complexes (LPcD) and Q-complexes (HLcPD) were crystal lamellas, whereas those of other nanocomplexes were spheres. Circular dichroism showed the changed configuration of peptide with spacer GA in nanocomplexes compared with that of its free state, whereas the Pa configuration without spacer in nanocomplexes was consistent with that of its free state. The present study contributed to the structural understanding of Q-complexes, and further effective modification is in progress.

The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations.

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes.

Multifunctional, self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery.

Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5' RACE-PCR and Western blot analysis of protein.

Polyspermine imine, a pH responsive polycationic siRNA carrier degradable to endogenous metabolites.

Cationic polymers readily degradable in response to cellular environment are especially favored as easy-formulating materials to pack siRNA into a nanoparticle and to release the cargo in the cytoplasm in time. In addition to the efficiency of cytosomal release, the degradation products should best be free of safety concerns, a typical challenge for cationic polymers. To satisfy the two criteria, we report a new cationic polymer, polyspermine imine, named as PSP-Imine, which is formed by condensing two endogenous molecules, spermine and glyoxal, through conjugated π linkage, -N═C-C═N- (Schiff base reaction), a poly linkage structure sufficiently stable under neutral condition but dissociative under the endosomal pH. Cellular assays under a confocal microscope indicated that the polyplex formed of PSP-Imine readily released the loaded siRNA to the cytoplasm after being engulfed in the target cells and efficiently silenced the target genes in different cell lines and xenograft mouse model of human cervical carcinoma, as compared with nondegradable PEI 25 kDa. Cell viability assays confirmed that PSP-Imine showed no visible cytotoxicity within the concentration being tested. The present study suggests that PSP-Imine is an excellent siRNA condensing material for forming the core of a therapeutically feasible synthetic carrier system.

Polymerized spermine as a novel polycationic nucleic acid carrier system.

Spermine, an endogenous amino-group bearing monomer that condenses DNA in sperm, was used as the basic building block to form polycationic nucleic acid carriers via condensation with one of three linker molecules - bischloroformate, succinyl chloride, and glyoxal. The three cationic polymers, polyspermine carbamate (PSP-Carb), polyspermine amide (PSP-Amide) and polyspermine imine (PSP-Imine) were examined for their degradability, cytotoxicity, ability to condense nucleic acids to nanoparticles, and ability to transfect genes or siRNA to cells. PSP-Carb and PSP-Amide exhibited a half-life of more than 2 months when incubated in aqueous buffers at 37°C, while the half-life of PSP-Imine was 11h. Relative cytotoxicity of the polymers, as measured by COS-7 and HepG2 cell viability, was in the order of PSP-Carb>PSP-Amide>PSP-Imine. Each cationic polymer condensed the luciferase plasmid to nanoparticles of 150-200 nm diameters and with a zeta potential of +15-30 mV when the mass ratio of polymer-to-DNA was over 8/1. The three polycationic carriers showed similar luciferase transfection activity in COS-7 cells, while the transfection efficiency of PSP-Carb was significantly higher than that of the other two in HepG2 cells. PSP-Amide exhibited significantly higher gene silencing activity in COS-7 cells, suggesting the linkage structures play an important role in the activity of the polyspermine-based nucleic acid carriers.