Zhi-Ting Cao

Tumor Acidity-Sensitive Polymeric Vector for Active Targeted siRNA Delivery.

Although surface PEGylation of siRNA vectors is effective for preventing protein adsorption and thereby helps these vectors to evade the reticuloendothelial system (RES) in vivo, it also suppresses the cellular uptake of these vectors by target cells. This dilemma could be overcome by employing stimuli-responsive shell-detachable nanovectors to achieve enhanced cellular internalization while maintaining prolonged blood circulation. Among the possible stimuli, dysregulated pH in tumor (pHe) is the most universal and practical. However, the design of pHe-sensitive system is problematic because of the subtle differences between the pHe and pH in other tissues. Here, a simple acid-sensitive bridged copolymer is developed and used for tumor-targeted systemic delivery of siRNA. After forming the micelleplex delivery system, the corresponding nanoparticles (Dm-NP) might undergo several modifications as follows: (i) a poly(ethylene glycol) (PEG) corona, which is stable in the circulatory system and protects nanovectors from RES clearance; (ii) a pHe responsive linkage breakage, which induces PEG detachment at tumor sites and thereby facilitates cell targeting; and (iii) a cell-penetration peptide, which is exposed upon the removal of PEG and further enhances cellular uptake. Thus, Dm-NP achieved both prolonged circulation and effective accumulation in tumor cells and resulted in the safe and enhanced inhibition of non-small cell lung cancer growth.

Facile Generation of Tumor‐pH‐Labile Linkage‐Bridged Block Copolymers for Chemotherapeutic Delivery

Successful bench-to-bedside translation of nanomedicine relies heavily on the development of nanocarriers with superior therapeutic efficacy and high biocompatibility. However, the optimal strategy for improving one aspect often conflicts with the other. Herein, we report a tactic of designing tumor-pH-labile linkage-bridged copolymers of clinically validated poly(D,L-lactide) and poly(ethylene glycol) (PEG-Dlink(m)-PDLLA) for safe and effective drug delivery. Upon arriving at the tumor site, PEG-Dlink(m)-PDLLA nanoparticles will lose the PEG layer and increase zeta potential by responding to tumor acidity, which significantly enhances cellular uptake and improves the in vivo tumor inhibition rate to 78.1% in comparison to 47.8% of the non-responsive control. Furthermore, PEG-Dlink(m)-PDLLA nanoparticles show comparable biocompatibility with the clinically used PEG-b-PDLLA micelle. The improved therapeutic efficacy and safety demonstrate great promise for our strategy in future translational studies.

Co-delivery of all-trans-retinoic acid and doxorubicin for cancer therapy with synergistic inhibition of cancer stem cells.

Combination treatment through simultaneous delivery of two or more drugs with nanoparticles has been demonstrated to be an elegant and efficient approach for cancer therapy. Herein, we employ a combination therapy for eliminating both the bulk tumor cells and the rare cancer stem cells (CSCs) that have a high self-renewal capacity and play a critical role in cancer treatment failure. All-trans-retinoic acid (ATRA), a powerful differentiation agent of cancer stem cells and the clinically widely used chemotherapy agent doxorubicin (DOX) are simultaneously encapsulated in the same nanoparticle by a single emulsion method. It is demonstrated that ATRA and DOX simultaneous delivery-based therapy can efficiently deliver the drugs to both non-CSCs and CSCs to differentiate and kill the cancer cells. Differentiation of CSCs into non-CSCs can reduce their self-renewal capacity and increase their sensitivity to chemotherapy; with the combined therapy, a significantly improved anti-cancer effect is demonstrated. Administration of this combinational drug delivery system can markedly augment the enrichment of drugs both in tumor tissues and cancer stem cells, prodigiously enhancing the suppression of tumor growth while reduce the incidence of CSC in a synergistic manner.

Macrophage-Specific in Vivo Gene Editing Using Cationic Lipid-Assisted Polymeric Nanoparticles

The CRISPR/Cas9 gene editing technology holds promise for the treatment of multiple diseases. However, the inability to perform specific gene editing in targeted tissues and cells, which may cause off-target effects, is one of the critical bottlenecks for therapeutic application of CRISPR/Cas9. Herein, macrophage-specific promoter-driven Cas9 expression plasmids (pM458 and pM330) were constructed and encapsulated in cationic lipid-assisted PEG-b-PLGA nanoparticles (CLAN). The obtained nanoparticles encapsulating the CRISPR/Cas9 plasmids were able to specifically express Cas9 in macrophages as well as their precursor monocytes both in vitro and in vivo. More importantly, after further encoding a guide RNA targeting Ntn1 (sgNtn1) into the plasmid, the resultant CLANpM330/sgNtn1 successfully disrupted the Ntn1 gene in macrophages and their precursor monocytes in vivo, which reduced expression of netrin-1 (encoded by Ntn1) and subsequently improved type 2 diabetes (T2D) symptoms. Meanwhile, the Ntn1 gene was not disrupted in other cells due to specific expression of Cas9 by the CD68 promoter. This strategy provides alternative avenues for specific in vivo gene editing with the CRISPR/Cas9 system.

Optimization of lipid-assisted nanoparticle for disturbing neutrophils-related inflammation

Inflammation is closely related to the development of many diseases and is commonly characterized by abnormal infiltration of immune cells, especially neutrophils. The current therapeutics of inflammatory diseases give little attention to direct modulation of these diseases with respect to immune cells. Nanoparticles are applied for efficient drug delivery into the disease-related immune cells, but their performance is significantly affected by their surface properties. In this study, to optimize the properties of nanoparticles for modulating neutrophils-related inflammation, we prepared a library of poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLANs) with different surface PEG density and surface charge. Optimized CLANs for neutrophils targeting were screened in high-fat diet (HFD)-induced type 2 diabetes (T2D) mice. Then, a CRISPR-Cas9 plasmid expressing a guide RNA (gRNA) targeting neutrophil elastase (NE) was encapsulated into the optimized CLAN and denoted as CLANpCas9/gNE. After intravenous injection, CLANpCas9/gNE successfully disrupted the NE gene of neutrophils and mitigated the insulin resistance of T2D mice via reducing the inflammation in epididymal white adipose tissue (eWAT) and in the liver. This strategy provides an example of abating the inflammatory microenvironment by directly modulating immune cells with nanoparticles carrying genome editing tools.

Targeting of NLRP3 inflammasome with gene editing for the amelioration of inflammatory diseases

The NLRP3 inflammasome is a well-studied target for the treatment of multiple inflammatory diseases, but how to promote the current therapeutics remains a large challenge. CRISPR/Cas9, as a gene editing tool, allows for direct ablation of NLRP3 at the genomic level. In this study, we screen an optimized cationic lipid-assisted nanoparticle (CLAN) to deliver Cas9 mRNA (mCas9) and guide RNA (gRNA) into macrophages. By using CLAN encapsulating mCas9 and gRNA-targeting NLRP3 (gNLRP3) (CLANmCas9/gNLRP3), we disrupt NLRP3 of macrophages, inhibiting the activation of the NLRP3 inflammasome in response to diverse stimuli. After intravenous injection, CLANmCas9/gNLRP3 mitigates acute inflammation of LPS-induced septic shock and monosodium urate crystal (MSU)-induced peritonitis. In addition, CLANmCas9/gNLRP3 treatment improves insulin sensitivity and reduces adipose inflammation of high-fat-diet (HFD)-induced type 2 diabetes (T2D). Thus, our study provides a promising strategy for treating NLRP3-dependent inflammatory diseases and provides a carrier for delivering CRISPR/Cas9 into macrophages.Activation of the NLRP3 inflammasome triggers the production of inflammatory cytokines. Here, the authors inactivate NLRP3 in macrophages using CRISPR/Cas9 encapsulated in nanoparticles, and show that administration in mice is effective in preventing septic shock and peritonitis, and in improving diabetes-associated inflammation and insulin resistance.

Simultaneous elimination of cancer stem cells and bulk cancer cells by cationic-lipid-assisted nanoparticles for cancer therapy

Convincing evidence indicates that the existence of cancer stem cells (CSCs) within malignant tumors is mostly responsible for the failure of chemotherapy. Therefore, instead of merely targeting bulk cancer cells, simultaneous elimination of both CSCs and bulk cancer cells is necessary to improve therapeutic outcomes. Herein, we designed cationic-lipid-assisted nanoparticles DTXLNPsiRNA for simultaneous encapsulation of the conventional chemotherapeutic agentdocetaxel (DTXL) and small interfering RNA (siRNA) targeting BMI-1 (siBMI-1). We confirmed that nanoparticles DTXLNPsiBMI-1 effectively deliver both therapeutic agents into CSCs and bulk cancer cells. The bulk cancer cells were effectively killed by the DTXL encapsulated in DTXLNPsiBMI-1. In breast CSCs, BMI-1 expression was significantly downregulated by DTXLNPsiBMI-1; consequently, the stemness was reduced and chemosensitivity of CSCs to DTXL was enhanced, resulting in the elimination of CSCs. Therefore, via DTXLNPsiBMI-1, the combination of siBMI-1 and DTXL completely inhibited tumor growth and prevented a relapse by synergistic killing of CSCs and bulk cancer cells in a murine model of an MDA-MB-231 orthotropic tumor.