Dexamethasone Pretreatment Potentiates a Folic Acid-Functionalized Delivery System for Enhanced Lung Cancer Therapy.

Folic acid (FA) has been widely engineered to promote the targeted delivery of FA-modified nanoparticles (NPs) by recognizing the folate receptor α (FRα). However, the efficacy of FA-targeted therapy significantly varied with the abundance of FRα and natural immunoglobulin levels in different tumors. Therefore, a sequential therapy of dexamethasone (Dex)-induced FRα amplification and immunosuppression combined with FA-functionalized doxorubicin (DOX) micelles to synergistically suppress tumor proliferation was proposed in this study. In brief, a pH/reduction-responsive FA-functionalized micelle (FCSD) was obtained by grafting FA, derivatization-modified cholesterol, and 2,3-dimethylmaleic anhydride onto a chitosan oligosaccharide. The obtained FCSD/DOX NPs can effectively deliver DOX in tumors, and their targeting efficiency can be further improved with Dex pretreatment to decrease the immunoglobulin M (IgM) content in serum and amplify FRα levels on the surface of M109 cells. After internalization, charge reversal and disulfide bond breakage of FCSD vectors under the stimulation of tumor extracellular pH (pHe) and intracellular glutathione (GSH) would contribute to the disintegration of vectors and the rapid release of DOX. The sequential therapy that combined Dex pretreatment and targeted chemotherapy by FCSD/DOX NPs demonstrated superior tumor suppression compared with monotherapy, which is expected to provide a potential strategy for FRα-positive lung cancer patients.

Steerable Microneedles Enabling Deep Delivery of Photosensitizers and CRISPR/Cas9 Systems for Effective Combination Cancer Therapy.

Although gene therapy has shown prospects in treating triple-negative breast cancer, it is insufficient to treat such a malignant tumor. Herein, nanoparticles (NPs)-embedded dissolving microneedles (IR780-PL/pFBXO44@MNs) with steerable and flectional property were developed to achieve the codelivery of FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and hydrophobic photosensitizers. For improved NP penetration in tumor tissue, collagenase@MNs were preapplied to degrade the tumor matrix. Under light irradiation, IR780 exhibited remarkable phototherapy, while the escape efficiency of NPs from lysosomes was improved. pFBXO44 was subsequently released in tumor cell cytoplasm via reducing the disulfide bonds of NPs, which could specifically knock out the FBXO44 gene to inhibit the migration and invasion of tumor cells. As a result, tumor cells were eradicated, and lung metastasis was effectively suppressed. This micelle-incorporated microneedle platform broadens the potential of combining gene editing and photo synergistic cancer therapy.

Tumor immunosuppression relief via acidity modulation combined PD-L1 siRNA for enhanced immunotherapy.

The efficacy of immune checkpoint therapy is limited by the immunosuppressive tumor microenvironment (TME), and lactate, the most universal component of TME, has been rediscovered that plays important roles in the regulation of metabolic pathways, angiogenesis, and immunosuppression. Here, a therapeutic strategy of acidity modulation combined with programmed death ligand-1 (PD-L1) siRNA (siPD-L1) is proposed to synergistically enhance tumor immunotherapy. The lactate oxidase (LOx) is encapsulated into the hollow Prussian blue (HPB) nanoparticles (NPs) prepared by hydrochloric acid etching followed by the modification with polyethyleneimine (PEI) and polyethylene glycol (PEG) via sulfur bonds (HPB-S-PP@LOx), siPD-L1 is loaded via electrostatic adsorption to obtain HPB-S-PP@LOx/siPD-L1. The obtained co-delivery NPs can accumulate in tumor tissue with stable systemic circulation, and simultaneous release of LOx and siPD-L1 in intracellular high glutathione (GSH) environment after uptake by tumor cells without being destroyed by lysosome. Moreover, LOx can catalyze the decomposition of lactate in the hypoxic tumor tissue with the aid of oxygen release by the HPB-S-PP nano-vector. The results show that the acidic TME regulation via lactate consumption can improve the immunosuppressive TME, including revitalizing the exhausted CD8+ T cells and decreasing the proportion of immunosuppressive Tregs, and synergistically elevating the therapeutic effect of PD1/PD-L1 blockade therapy via siPD-L1. This work provides a novel insight for tumor immunotherapy and explores a promising therapy for triple-negative breast cancer.

Across-cancer specific immune responses induced by nanovaccines or microvaccines to prevent different cancers and cancer metastasis.

Metastatic cancers and recurrent cancers are diverse, different from primary cancers, and organ-dependent. However, how strong are across-cancer immune responses among different types of cancers remain unclear. Herein, vaccines-encapsulated-whole-components-of-tumor-tissue (VEWCOTT) were applied to demonstrate the across-cancer immune responses, thanks to inducing pan-clones T-cell immune responses. Either lung-cancer-tissue- or melanoma-tissue-based VEWCOTT simultaneously prevented melanoma, lung cancer, hepatoma, and metastatic cancer, which showed that strong across-cancer immune responses were induced. Both nanovaccines and microvaccines showed potent across-cancer prevention efficacy. VEWCOTT induced tumor-specific T cells in peripheral immune organs and major organs, and adjusted the immune-microenvironment of cancer-colonized organs. In addition, the allograft of T cells from VEWCOTT immunized mice to allogeneic naive mice efficiently prevent various cancers. Many neoantigens are shared by melanoma cells and lung cancer cells. Across-cancer immune responses exist among different types of cancers, and thus VEWCOTT has the advantage of simultaneously preventing cancer metastasis and cancers in different organs.

A multifunctional non-viral vector for the delivery of MTH1-targeted CRISPR/Cas9 system for non-small cell lung cancer therapy.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.

Coadministration of iRGD peptide with ROS-sensitive nanoparticles co-delivering siFGL1 and siPD-L1 enhanced tumor immunotherapy.

The continuous activation and expansion of tumor-specific T cells by various means are the main goal of cancer immunotherapy. Tumor cells overexpress fibrinogen-like protein 1 (FGL1) and programmmed death-ligand 1 (PD-L1), which respectively bind to lymphocyte-activation gene 3 (LAG-3) and programmmed death-1(PD-1) on T cells, forming important signaling pathways (FGL1/LAG-3 and PD-1/PD-L1) that negatively regulate immune responses. In order to interfere with the inhibitory function of FGL1 and PD-L1 proteins, we designed a new type of reactive oxygen species (ROS)-sensitive nanoparticles to load FGL1 siRNA (siFGL1) and PD-L1 siRNA (siPD-L1), which was formed from a stimuli-responsive polymer with a poly-l-lysine-thioketal and modified cis-aconitate to facilitate endosomal escape. Moreover, tumor-penetrating peptide iRGD and ROS-responsive nanoparticles were co-administered to further enhance the delivery efficiency of siFGL1 and siPD-L1, thereby significantly reducing the protein levels of FGL1 and PD-L1 in tumor cells. Our findings indicated that the dual delivery of FGL1/PD-L1 siRNA was a new and powerful treatment method, which was characterized by increasing the infiltration of effector CD4+ and CD8+ T cells, effectively alleviating the tumor immunosuppressive microenvironment. These findings also supported the superiority and feasibility of nanoparticle-mediated tumor immunotherapy, and may provide a different perspective for cancer treatment. STATEMENT OF SIGNIFICANCE: In addition to the idea that cancer vaccines can promote T cell immune responses, nanoparticle delivery modulators (such as small interfering RNA (siRNA) targeting immunosuppressive pathways) may provide more information for the research of nanoparticle-mediated cancer immunotherapy. In this study, we designed a new intelligent nano-delivery system for co-delivery of siFGL1 and siPD-L1, and demonstrated the ability to down-regulate the expression levels of FGL1 and PD-L1 proteins in tumor cells in vitro and in vivo. The constructed nanoparticle had a good tumor microenvironment responsiveness, and the delivery efficiency was enhanced by co-injection with tumor penetrating peptide iRGD. This project proposed a new strategy for tumor immunotherapy based on smart nano-delivery systems, and explored more possibilities for tumor therapy.

Immunotherapy and Prevention of Cancer by Nanovaccines Loaded with Whole-Cell Components of Tumor Tissues or Cells.

Tumor tissues/cells are the best sources of antigens to prepare cancer vaccines. However, due to the difficulty of solubilization and delivery of water-insoluble antigens in tumor tissues/cells, including water-insoluble antigens into cancer vaccines and delivering such vaccines efficiently to antigen-presenting cells (APCs) remain challenging. To solve these problems, herein, water-insoluble components of tumor tissues/cells are solubilized by 8 m urea and thus whole components of micrometer-sized tumor cells are reasssembled into nanosized nanovaccines. To induce maximized immunization efficacy, various antigens are loaded both inside and on the surface of nanovaccines. By encapsulating both water-insoluble and water-soluble components of tumor tissues/cells into nanovaccines, the nanovaccines are efficiently phagocytosed by APCs and showed better therapeutic efficacy than the nanovaccine loaded with only water-soluble components in melanoma and breast cancer. Anti-PD-1 antibody and metformin can improve the efficacy of nanovaccines. In addition, the nanovaccines can prevent lung cancer (100%) and melanoma (70%) efficiently in mice. T cell analysis and tumor microenvironment analysis indicate that tumor-specific T cells are induced by nanovaccines and both adaptive and innate immune responses against cancer cells are activated by nanovaccines. Overall, this study demonstrates a universal method to make tumor-cell-based nanovaccines for cancer immunotherapy and prevention.

Lactate Consumption via Cascaded Enzymes Combined VEGF siRNA for Synergistic Anti-Proliferation and Anti-Angiogenesis Therapy of Tumors.

Lactate, as the most abundant component with concentrations of 4-40 mm in tumors, contributes to the regulation of metabolic pathways, angiogenesis, and immunosuppression, exhibiting remarkable potential in cancer treatment. Therefore, a codelivery strategy that combined the cascaded enzymes Lactate oxidase/Catalase (LOx/CAT) and vascular endothelial growth factor (VEGF) siRNA (siVEGF) to suppress tumor proliferation and angiogenesis synergistically is creatively proposed. In brief, the cationic liposomes (LIP) encapsulated with LOx/CAT and siVEGF via hydrophilic interaction and electrostatic adsorption followed by coating with PEGylated phenylboronic acid (PP) is established (PPL@[LOX+CAT]). Moreover, a simple 3-aminophenylboronic acid (PBA)-shielded strategy via fructose (Fru) is applied to further enhance the targeting efficiency in the tumor site. The obtained co-encapsulated nanoparticles (NPs) can simultaneous intracellular release of LOx/CAT and siVEGF, and the collaborative use of LOx and CAT can promote lactate consumption even under a hypoxic tumor microenvironment (TME) without producing systemic toxicity. The combined application of lactate depletion and VEGF silencing demonstrated the efficient migration suppression of 4T1 cells in vitro and superior antitumor and antimetastatic properties in vivo. This work offers a promising tumor treatment strategy via integrating cascaded enzymes and gene therapy, and explores a promising therapy regimen for 4T1 triple-negative breast cancer.

Advances in receptor modulation strategies for flexible, efficient, and enhanced antitumor efficacy.

Tumor-sensitivity, effective transport, and precise delivery to tumor cells of nano drug delivery systems (NDDs) have been great challenges to cancer therapy in recent years. The conventional targeting approach involves actively installing the corresponding ligand on the nanocarriers, which is prone to recognize the antigen blasts overexpressed on the surface of tumor cells. However, there are some probable limitations for the active tumor-targeting systems in vivo as follows: a. the limited ligand amount of modifications; b. possible steric hindrance, which was likely to prevent ligand-receptor interaction during the delivery process. c. the restrained antigen saturation highly expressed on the cell membrane, will definitely decrease the specificity and often lead to "off-target" effects of NDDs; and d. water insolubility of nanocarriers due to excess of ligands modification. Obviously, any regulation of receptors on surface of tumor cells exerted an important influence on the delivery of targeting systems. Herein, receptor upregulation was mostly desired for enhancing targeted therapy from the cellular level. This technique with the amplification of receptors has the potential to enhance tumor sensitivity towards corresponding ligand-modified nanoparticles, and thereby increasing the effective therapeutic concentration as well as improving the efficacy of chemotherapy. The enhancement of positively expressed receptors on tumor cells and receptor-dependent therapeutic agents or NDDs with an assembled "self-promoting" effect contributes to increasing cell sensitivity to NPs, and will provide a basic platform for clinical therapeutic practice. In this review, we highlight the significance of modulating various receptors on different types of cancer cells for drug delivery and therapeutic benefits.

Efficient tumor-targeting delivery of siRNA via folate-receptor mediated biomimetic albumin nanoparticles enhanced by all-trans retinoic acid.

Acute myeloid leukemia (AML) is the most universal type and fatal disease of hematological malignancy, with poor outcomes despite chemotherapy and bone marrow transplantations. Benefited from the narrow tissue specificity of folate receptor ß (FRß) aberrantly expressed on hematological linage cell lines, NPs modified with folate acid (FA) has been widely applied for crossing cell membrane barriers in FR-targeted therapies for AML. Thus, the biomimetic nanoparticles (NPs) mediated by FRß were conducted by an albumin modifier as previously synthesized and cationic liposomes. However, how to further enhance the tumor-targeting and cellular uptake of NPs have been great challenges in cancer therapy. It was reported that FRß could be selectively augmented by all-trans retinoic acid (ATRA). Herein, we demonstrated the enhanced active tumor-targeting of FA-modified siRNA-loaded biomimetic albumin NPs (Lip-S@FBH) could be achieved by upregulating FRß expression via ATRA NPs. And the systematic administration of ATRA NPs significantly promoted endocytosis and thereby increased the intracellular concentration of Lip-S@FBH. This strategy combined the FRß amplification effect with the effective delivery of siRNA, is mostly desirable for the AML-targeting therapy.

Folate-receptor mediated pH/reduction-responsive biomimetic nanoparticles for dually activated multi-stage anticancer drug delivery.

How to overcome the cell membrane barriers and achieve release payloads efficiently in the cytoplasm have been major challenges for anticancer drug delivery and therapeutic effects with nanosystems. In this study, bovine serum albumin (BSA) was modified with folate acid and histamine, which was then used as the nanocarrier for the antitumor agent doxorubicin (DOX). The DOX-loaded nanoparticles (DOX/FBH-NPs) were prepared via a crosslinking method, and the release of DOX from these nanoparticles (NPs) exhibited pH/reduction-responsive behaviors in vitro. These NPs interacted with the folate receptor overexpressed on the cell membrane of 4 T1 cells and achieved enhanced endocytosis. Afterwards, these NPs exhibited pH-responsiveness within endo-lysosomes and escaped from endosomes due to the "proton sponge" effect, and then completed release of DOX was triggered by high concentration of glutathione (GSH) in cytoplasm. Thus, DOX/FBH-NPs exhibited excellent cytotoxicity against 4 T1 cells in vitro. Benefited from the enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis, these NPs gained satisfied tumor-targeting effects in vivo and efficient delivery of DOX to tumor tissues. As a result, these NPs exhibited enhanced antitumor effects and reduced side effects in vivo. In conclusion, these BSA-based NPs modified with both folate acid and histamine showed enhanced tumor-targeting effects in vivo with good biocompatibility and intracellular pH/reduction-responsive behaviors, providing a promising strategy for the efficient delivery of antitumor agents.

Stimuli-responsive nanoparticles for the codelivery of chemotherapeutic agents doxorubicin and siPD-L1 to enhance the antitumor effect.

Cancer cells have been reported to exhibit high resistance against immune system recognition through various cell intrinsic and extrinsic mechanisms. Considerable challenges have been encountered in monotherapy with chemotherapeutics to attain the desired antitumor efficacy. In this study, a nanodelivery system was designed to incorporate doxorubicin (DOX) and programmed death-ligand 1 (PD-L1) small interfering RNA (siRNA), that is, siPD-L1. DOX and siPD-L1 were formed from a stimuli-responsive polymer with a poly-L-lysine-lipoic acid reduction-sensitive core and a tumor extracellular pH-stimulated shedding polyethylene glycol layer. The codelivery system was stable under physiological pH conditions and demonstrated enhanced cellular uptake at the tumor site. Moreover, the combined treatment of DOX and siPD-L1 exhibited improved antitumor effect in vitro and in vivo compared with either modality alone. The combination of chemotherapy and immunotherapy presented in this work through the codelivery of a chemotherapeutic agent and a gene-silencing agent (siRNA) may provide a new strategy for cancer treatment.

Co-delivery of VEGF siRNA and Etoposide for Enhanced Anti-angiogenesis and Anti-proliferation Effect via Multi-functional Nanoparticles for Orthotopic Non-Small Cell Lung Cancer Treatment.

Targeting tumor angiogenesis pathway via VEGF siRNA (siVEGF) has shown great potential in treating highly malignant and metastatic non-small cell lung cancer (NSCLC). However, anti-angiogenic monotherapy lacked sufficient antitumor efficacy which suffered from malignant tumor proliferation. Therefore, the combined application of siVEGF and chemotherapeutic agents for simultaneous targeting of tumor proliferation and angiogenesis has been a research hotspot to explore a promising NSCLC therapy regimen. Methods: We designed, for the first time, a rational therapy strategy via intelligently co-delivering siVEGF and chemotherapeutics etoposide (ETO) by multi-functional nanoparticles (NPs) directed against the orthotopic NSCLC. These NPs consisted of cationic liposomes loaded with siVEGF and ETO and then coated with versatile polymer PEGylated histidine-grafted chitosan-lipoic acid (PHCL). We then comprehensively evaluated the anti-angiogenic and anti-proliferation efficiency in the in vitro tumor cell model and in bioluminescent orthotopic lung tumor bearing mice model. Results: The NPs co-delivering siVEGF and ETO exhibited tailor-made surface charge reversal features in mimicking tumor extracellular environment with improved internal tumor penetration capacity and higher cellular internalization. Furthermore, these NPs with flexible particles size triggered by intracellular acidic environment and redox environment showed pinpointed and sharp intracellular cargo release guaranteeing adequate active drug concentration in tumor cells. Enhanced VEGF gene expression silencing efficacy and improved tumor cell anti-proliferation effect were demonstrated in vitro. In addition, the PHCL layer improved the stability of these NPs in neutral environment allowing enhanced orthotopic lung tumor targeting efficiency in vivo. The combined therapy by siVEGF and ETO co-delivered NPs for orthotopic NSCLC simultaneously inhibited tumor proliferation and tumor angiogenesis resulting in more significant suppression of tumor growth and metastasis than monotherapy. Conclusion: Combined application of siVEGF and ETO by the multi-functional NPs with excellent and on-demand properties exhibited the desired antitumor effect on the orthotopic lung tumor. Our work has significant potential in promoting combined anti-angiogenesis therapy and chemotherapy regimen for clinical NSCLC treatment.

Doxorubicin and siRNA-PD-L1 co-delivery with T7 modified ROS-sensitive nanoparticles for tumor chemoimmunotherapy.

Tumor cells avoid immunosurveillance during the tumorigenesis, metastasis and recurrence periods thanks to the overexpressed immunosuppressive molecules on their surface. For instance, the programmed cell death 1 ligand (PD-L1) binds with the T-cells' programmed cell death receptor 1 (PD-1) impairing the anti-tumor activity of the host T cells. In this study, a new reactive oxygen species (ROS) responsive nanoparticle (NP), modified with the HAIYPRH (T7) peptide, was developed for the co-delivery of siRNA-PD-L1 and doxorubicin (Dox). These NPs can block the inhibitory signal responding to T cells and enhance cytotoxicity of Dox against tumor cells. The T7 modification binds to the overexpressed transferrin receptor on tumor cells facilitating its cellular uptake. Dox rapid release is then triggered by the high tumor cells cytoplasmic concentration of ROS, leading to cell apoptosis. Our results demonstrated these NPs exhibited a T7-mediated cellular uptake and an intracellular ROS-triggered payloads release in vitro. They also suggested an improved in vivo 4T1 tumor targeting efficiency and chemoimmunotherapy. Most notably, the co-delivery system exhibited a significantly enhanced antitumor effect over Dox-only loaded NPs following prompting the proliferation of T cells by siRNA-PD-L1. In conclusion, these ROS-responsive NPs provided a promising strategy to combine siRNA-PD-L1 immunotherapy and Dox chemotherapy.

A Novel 3D in Vitro Tumor Model Based on Silk Fibroin/Chitosan Scaffolds To Mimic the Tumor Microenvironment.

Drug development involves various evaluation processes to ascertain drug effects and rigorous analysis of biological indicators during in vitro preclinical studies. Two-dimensional (2D) cell cultures are commonly used in numerous in vitro studies, which are poor facsimiles of the in vivo conditions. Recently, three-dimensional (3D) tumor models mimicking the tumor microenvironment and reducing the use of experimental animals have been developed generating great interest to appraise tumor response to treatment strategies in cancer therapy. In this study, silk fibroin (SF) protein and chitosan (CS), two natural biomaterials, were chosen to construct the scaffolds of 3D cell models. Human non-small cell lung cancer A549 cells in the SF/CS scaffolds were found to have a great tendency to gather and form tumor spheres. A549 cell spheres in the 3D scaffolds showed biological and morphological characteristics much closer to the in vivo tumors. Besides, the cells in 3D models displayed better invasion ability and drug resistance than 2D models. Additionally, differences in drug-resistant and immune-related protein levels were found, which indicated that 3D models might resemble the real-life situation. These findings suggested that these 3D tumor models composed of SF/CS are promising to provide a valuable biomaterial platform in the evaluation of anticancer drugs.

Programmed pH/reduction-responsive nanoparticles for efficient delivery of antitumor agents in vivo.

To bypass the biological barriers during the drug delivery process, it is desirable to develop smart nanoparticles (NPs) with flexible physical and chemical properties. In this study, a programmed NP delivery system with a pH-triggered detachable PEG layer and a lactobionic acid (Lac)-modified reduction-responsive core was developed to address the "PEG dilemma" and provide an on-demand intracellular release of doxorubicin (DOX). The positively charged DOX-loaded lactobionic acid-chitosan-lipoic acid (DOX/LCL) NPs were prepared and coated with a negatively charged dimethylmaleic acid-PEG-chitosan (PEG-CS-DA) layer to obtain a prolonged circulation time and improve the tumor-targeting effect. After reaching the tumor tissues through a targeted delivery effect, the surface charge of the PEG-CS-DA layer was reversed from negative to positive because of the trigger by the acidic microenvironment (pH 6.8), thus leading to the detachment of the PEG layer. The exposure of positive charges and the active targeting ligand enhanced cellular uptake and facilitated penetration into tumor tissues. Subsequently, the rapid release and diffusion of DOX into the nuclei was triggered by the intracellular high concentration of glutathione, thus leading to cell apoptosis. In conclusion, these programmed pH/reduction-responsive NPs provide a promising strategy for the delivery of antitumor agents in vivo. STATEMENT OF SIGNIFICANCE: In this study, novel programmed pH/reduction-responsive NPs were developed for the delivery of DOX in vivo. These NPs were coated with a negatively charged PEG layer to improve the serum stability and tumor target effect. The PEG layer detached because of the trigger by tumor acidic microenvironment (pH 6.8), thus leading to the exposure of positive charges and the active targeting ligand, which enhanced cellular uptake and facilitated penetration into tumor tissues. Subsequently, the rapid release of DOX was triggered by the intracellular high concentration of glutathione, thereby resulting in enhanced cytotoxicity. These programmed pH/reduction-responsive NPs provide a promising strategy for the delivery of antitumor agents in vivo.

Targeted Delivery of Doxorubicin via CD147-Mediated ROS/pH Dual-Sensitive Nanomicelles for the Efficient Therapy of Hepatocellular Carcinoma.

Low accumulation in tumor sites and slow intracellular drug release remain as the obstacles for nanoparticles to achieve effective delivery of chemotherapeutic drugs. In this study, multifunctional micelles were designed to deliver doxorubicin (Dox) to tumor sites to provide more efficient therapy against hepatic carcinoma. The micelles were based on pH-responsive carboxymethyl chitosan (CMCh) modified with a reactive oxygen species (ROS)-responsive segment phenylboronic acid pinacol ester (BAPE) and an active targeted ligand CD147 monoclonal antibody. The Dox-loaded micelles provided rapid and complete drug release in pH 5.3 incubation conditions with 1 mM H2O2. In addition, an in vitro cell uptake study revealed that CD147 modification significantly enhanced cellular internalization due to the high affinity to CD147 receptors, which are overexpressed on tumor cells. An in vivo study revealed that CD147-modified micellar formulations exhibited high accumulation in tumor sites and markedly enhanced antiproliferation effects with fewer side effects than other formulations. In conclusion, this CD147 receptor targeted delivery system with ROS/pH dual sensitivity provides a promising strategy for the treatment of hepatic carcinoma.

Nucleolin-Targeting AS1411-Aptamer-Modified Graft Polymeric Micelle with Dual pH/Redox Sensitivity Designed To Enhance Tumor Therapy through the Codelivery of Doxorubicin/TLR4 siRNA and Suppression of Invasion.

In this article, a novel graft polymeric micelle with targeting function ground on aptamer AS1411 was synthesized. The micelle was based on chitosan-ss-polyethylenimine-urocanic acid (CPU) with dual pH/redox sensitivity and targeting effects. This micelle was produced for codelivering Toll-like receptor 4 siRNA (TLR4-siRNA) and doxorubicin (Dox). In vitro investigation revealed the sustained gene and drug release from Dox-siRNA-loaded micelles under physiological conditions, and this codelivery nanosystem exhibited high dual pH/redox sensitivity, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Furthermore, the micelles loaded with TLR4-siRNA inhibited the migration and invasion of A549. Excellent tumor penetrating efficacy was also noted in the A549 tumor spheroids and solid tumor slices. In vivo, multiple results demonstrated the excellent tumor-targeting ability of AS1411-chitosan-ss-polyethylenimine-urocanic acid (ACPU) micelle in tumor tissues. The micelles exhibited excellent antitumor efficacy and low toxicity in the systemic circulation in lung-tumor-bearing BALB/c mice. These results conclusively demonstrated the great potential of the new graft copolymer micelle with targeting function for the targeted and efficient codelivery of chemotherapeutic drugs and genes in cancer treatment.

Stepwise pH/reduction-responsive polymeric conjugates for enhanced drug delivery to tumor.

In this research, a charge-conversional polymer, poly-l-lysine-lipoic acid (PLL-LA), was prepared by dimethylmaleic anhydride (DA) modification and applied as a carrier with enhanced cell internalization and intracellular pH- and reduction-triggered doxorubicin (Dox) release. The surface charge of dimethylmaleic anhydride-poly-l-lysine-lipoic acid micelles (DA-PLL-LA) was negative at physiological pH and reversed to positive at the extracellular and intracellular pH of cancer cells. At tumor extracellular pH of 6.8, the conjugates underwent a rapid charge-reversible process with almost 80% DA cleavage within 2h, and then endocytosed into the endo/lysosomes more rapidly than at physiological pH of 7.4. The Dox/DA-PLL-LA micelles (Dox-micelles) demonstrated a sustained drug release in vitro under physiological condition, and rapid Dox release was triggered by both extracellular pH and high-concentration reducing glutathione. The Dox-micelles also exhibited enhanced internalization at extracellular pH, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Excellent tumor-penetrating efficacy was also found in A549 tumor spheroids and solid tumor slices. Moreover, the DA-PLL-LA micelles exhibited excellent tumor-targeting ability in tumor tissues and excellent antitumor efficacy and low systemic toxicity in breast tumor-bearing mice. Therefore, the DA-PLL-LA micelles demonstrated great potential for targeted and efficient drug delivery in cancer treatments.

Multifunctional nanoparticles co-delivering EZH2 siRNA and etoposide for synergistic therapy of orthotopic non-small-cell lung tumor.

Malignant proliferation and metastasis in non-small cell lung carcinoma (NSCLC) are great challenges for effective clinical treatment through conventional chemotherapy. The combinational therapy strategy of RNA interfering (RNAi) technology and chemotherapeutic agents have been reported to be promising for effective cancer therapy. In this study, based on multifunctional nanoparticles (NPs), the simultaneous delivery of etoposide (ETP) and anti-Enhancer of Zeste Homologue 2 (EZH2) siRNA for the effective treatment of orthotopic lung tumor was achieved. The NPs exhibited pH/redox dual sensitivity verified by particle size changes, morphological changes, and in vitro release of drugs. Confocal microscopy analysis confirmed that the NPs exhibited endosomal escape property and on-demand intracellular drug release behavior, which can protect siRNA from degradation and facilitate the chemotherapeutic effect respectively. In vitro tumor cell motility study demonstrated that EZH2 siRNA loaded in NPs can decrease the migration and invasion capabilities of tumor cells by downregulating the expression of EZH2 mRNA and protein. In particular, an antiproliferation study revealed that the co-delivery of siRNA and ETP in the multifunctional NPs can induce a synergistic therapeutic effect on NSCLC. In vivo targeting evaluation showed that cRGDyC-PEG modification on NPs exhibited a low distribution in normal organs and an obvious accumulation in orthotopic lung tumor. Furthermore, targeted NPs co-delivering siRNA and ETP showed superior inhibition on tumor growth and metastasis and produced minimal systemic toxicity. These findings indicated that multifunctional NPs can be utilized as a co-delivery system, and that the combination of EZH2 siRNA and ETP can effectively treat NSCLC.