Ying-Li Luo

Regulating the Surface Poly(ethylene glycol) Density of Polymeric Nanoparticles and Evaluating its Role in Drug Delivery in vivo

Poly(ethylene glycol) (PEG) is usually used to protect nanoparticles from rapid clearance in blood. The effects are highly dependent on the surface PEG density of nanoparticles. However, there lacks a detailed and informative study in PEG density and inxa0vivo drug delivery due to the critical techniques to precisely control the surface PEG density when maintaining other nano-properties. Here, we regulated the polymeric nanoparticles size and surface PEG density by incorporating poly(ε-caprolactone) (PCL) homopolymer into poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) and adjusting the mass ratio of PCL to PEG-PCL during the nanoparticles preparation. We further developed a library of polymeric nanoparticles with different but controllable sizes and surface PEG densities by changing the molecular weight of the PCL block in PEG-PCL and tuning the molar ratio of repeating units of PCL (CL) to that of PEG (EG). We thus obtained a group of nanoparticles with variable surface PEG densities but with other nano-properties identical, and investigated the effects of surface PEG densities on the biological behaviors of nanoparticles in mice. We found that, high surface PEG density made the nanoparticles resistant to absorption of serum protein and uptake by macrophages, leading to a greater accumulation of nanoparticles in tumor tissue, which recuperated the defects of decreased internalization by tumor cells, resulting in superior antitumor efficacy when carrying docetaxel.

Promoting tumor penetration of nanoparticles for cancer stem cell therapy by TGF-β signaling pathway inhibition

Cancer stem cells (CSCs), which hold a high capacity for self-renewal, play a central role in the development, metastasis, and recurrence of various malignancies. CSCs must be eradicated to cure instances of cancer; however, because they can reside far from tumor vessels, they are not easily targeted by drug agents carried by nanoparticle-based drug delivery systems. We herein demonstrate that promoting tumor penetration of nanoparticles by transforming growth factor β (TGF-β) signaling pathway inhibition facilitates CSC therapy. In our study, we observed that although nanoparticles carrying siRNA targeting the oncogene polo-like kinase 1 (Plk1) efficiently killed breast CSCs derived from MDA-MB-231xa0cells inxa0vitro, this intervention enriched CSCs in the residual tumor tissue following systemic treatment. However, inhibition of the TGF-β signaling pathway with LY364947, an inhibitor of TGF-β type I receptor, promoted the penetration of nanoparticles in tumor tissue, significantly ameliorating the intratumoral distribution of nanoparticles in MDA-MB-231 xenografts and further leading to enhanced internalization of nanoparticles by CSCs. As a result, synergistic treatment with a nanoparticle drug delivery system and LY364947 inhibited tumor growth and reduced the proportion of CSCs inxa0vivo. This study suggests that enhanced tumor penetration of drug-carrying nanoparticles can enhance CSCs clearance inxa0vivo and consequently provide superior anti-tumor effects.

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.

Acidity-triggered TAT-presenting nanocarriers augment tumor retention and nuclear translocation of drugs

Hierarchical targeting strategy can combat the sequential drug delivery barriers by changing their properties with response to tumor stimuli. Among these strategies, much less attention has been paid to address the issues of rapid tumor clearance and insufficient cellular translocation. In this work, we demonstrate that a transactivator of transcription (TAT)-presenting nanomedicine (DATAT-NP/Pt), apart from improving tumor accumulation and cellular uptake, can simultaneously enhance tumor retention and promote nuclear translocation of encapsulated platinum prodrugs, and thus improve therapeutic efficacy. Specifically, a protecting 2,3-dimethylmaleic anhydride (DA) corona on the nanomedicine prevented the TAT peptide from serum. DATAT-NP/Pt efficiently accumulated at the tumor site through the enhanced permeability and retention (EPR) effect, followed by acid-triggered TAT presenting within the tumor acidic microenvironment (pH ~ 6.8). The exposed TAT peptide augmented tumor retention and nuclear translocation of DATAT-NP/Pt. We used a tumor-on-a-chip microfluidic system to real-time mimic and analyze tumor accumulation and retention at physiological flow conditions and revealed that surface absorption of nanomedicines on tumors was critical in determining their tumor retention and clearance. Furthermore, the TAT peptide rapidly translocated the DATAT-NP/Pt into the perinuclear region, allowing for higher nuclear platinum concentrations and increased Pt-DNA adduct formation in nuclei, which consequently reversed cisplatin resistance. Our work presents a new strategy to overcome pathophysiological barriers of tumor clearance and insufficient cellular translocation and provides new insights for the design of cancer nanomedicines.

Cationic Polymeric Nanoparticle Delivering CCR2 siRNA to Inflammatory Monocytes for Tumor Microenvironment Modification and Cancer Therapy

Accumulating evidence has confirmed that malignant tumors have a complex microenvironment, which consists of a heterogeneous collection of tumor cells and other cell subsets (including the full gamut of immune cells). Tumor-associated macrophages (TAMs), derived from circulating Ly6Chi monocytes, constitute the most substantial fraction of tumor-infiltrating immune cells in nearly all cancer types and contribute to tumor progression, vascularization, metastasis, immunosuppression, and therapeutic resistance. Interrupting monocyte recruitment to tumor tissues by disturbing pivotal signaling pathways (such as CCL2-CCR2) is viewed as one of the most promising avenues for tumor microenvironment manipulation and cancer therapy. One critical issue for monocyte-based therapy is to deliver therapeutic agents into monocytes efficiently. In the present study, we systematically investigated the relationship between the surface potential and the biodistribution of polymeric nanoparticles in monocytes in vivo, aiming to screen and identify an appropriate delivery system for monocyte targeting, and we found that cationic nanoparticles have a higher propensity to accumulate in monocytes compared with their neutral counterparts. We further demonstrated that siCCR2-encapsulated cationic nanoparticle (CNP/siCCR2) could modify immunosuppressive tumor microenvironment more efficiently and exhibit superior antitumor effect in an orthotopic murine breast cancer model.

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.

Optimized nanoparticle-mediated delivery of CRISPR-Cas9 system for B cell intervention

B cells exert multiple effector functions, and dysfunctions of B cells often lead to initiation and progression of diseases, including autoimmune and inflammatory diseases. Therefore, B cell intervention may be an effective strategy to treat diseases involving B cells. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing system has been widely used for DNA deletion, insertion, and replacement. Nanocarriers have been developed as relatively mature systems and may be applied to deliver the CRISPR-Cas9 system to B cells in vivo. In this study, we created a library of nanoparticles (NPs) with different polyethylene glycol densities and zeta potentials and screened an optimal NP for in vivo B cell targeting. The selected NP could deliver the CRISPR-Cas9 system to B cells and induce Cas9 expression inside the cell environment. Injection of the NP encapsulated with Cas9/gB220 (NPCas9/gB220) into mice could disrupt B220 expression in B cells, suggestive of its applications to intervene the expression of the target molecule in B cells. Moreover, the treatment with NPCas9/gBAFFR could decrease the number of B cells and exert therapeutic effect in rheumatoid arthritis, as B-cell activating factor receptor (BAFFR) is vital for the survival and functions of B cells. In conclusion, we developed a carrier for the delivery of the CRISPR-Cas9 gene editing system for B cell intervention that could be used for the treatment of diseases related to B cell dysfunctions.

The effect of surface poly(ethylene glycol) length on in vivo drug delivery behaviors of polymeric nanoparticles

Engineering nanoparticles of reasonable surface poly(ethylene glycol) (PEG) length is important for designing efficient drug delivery systems. Eliminating the disturbance by other nanoproperties, such as size, PEG density, etc., is crucial for systemically investigating the impact of surface PEG length on the biological behavior of nanoparticles. In the present study, nanoparticles with different surface PEG length but similar other nanoproperties were prepared by using poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) copolymers of different molecular weights and incorporating different contents of PCL3500 homopolymer. The molecular weight of PEG block in PEG-PCL was between 3400 and 8000u202fDa, the sizes of nanoparticles were around 100u202fnm, the terminal PEG density was controlled at 0.4 PEG/nm2 (or the frontal PEG density was controlled at 0.16 PEG/nm2). Using these nanoproperties well-designed nanoparticles, we demonstrated PEG length-dependent changes in the biological behaviors of nanoparticles and exhibited nonmonotonic improvements as the PEG molecular weight increased from 3400 to 8000u202fDa. Moreover, under the experimental conditions, we found nanoparticles with a surface PEG length of 13.8u202fnmu202f(MWu202f=u202f5000u202fDa) significantly decreased the absorption with serum protein and interaction with macrophages, which led to prolonged blood circulation time, enhanced tumor accumulation and improved antitumor efficacy. The present study will help to establish a relatively precise relationship between surface PEG length and the in vivo behavior of nanoparticles.