Research Article Dynamically crosslinked nanocapsules for the efficient and serumresistant cytosolic protein delivery

Intracellular protein delivery is critical to the development of protein-based biopharmaceuticals and therapies.However,current delivery vectors often suffer from complicated syntheses,low generality among various proteins,and insufficient serum stability.Herein,we developed an enlightened cytosolic protein delivery strategy by dynamically crosslinking epigallocatechin gallate(EGCG),low-molecular-weight polyethylenimine(PEI 1.8k),and 2-acetylphenylboric acid(2-APBA)on the protein surface,hence forming the EPP-protein nanocapsules(NCs).EGCG enhanced protein encapsulation via hydrogen bonding,and reduced the positive charge density of PEI to endow the NCs with high serum tolerance,thereby enabling effective cellular internalization in serum.The formation of reversible imine and boronate ester among 2-APBA,EGCG,and PEI 1.8k allowed acid-triggered dissociation of EPP-protein NCs in the endolysosomes,which triggered efficient intracellular release of the native proteins.Such strategy therefore showed high efficiency and universality for diversities of proteins with different molecular weights and isoelectric points,including enzyme,toxin,antibody,and CRISPR(clustered regularly interspaced short palindromic repeats)-Cas9 ribonucleoprotein(RNP),outperforming the commercial protein transduction reagent PULSin and RNP transfection reagent lipofectamine CMAX.Moreover,intravenously(i.v.)injected EPP-saporin NCs efficiently delivered saporin into 4T1 tumor cells to provoke robust antitumor effect.This simple,versatile,and robust cytosolic protein delivery system holds translational potentials for the development of protein-based therapeutics.

STX5 Inhibits Hepatocellular Carcinoma Adhesion and Promotes Metastasis by Regulating the PI3K/mTOR Pathway

Background and Aims:Syntaxin 5(STX5)is a member of the syntaxin or target-soluble SNAP receptor(t-SNARE)fam-ily and plays a critical role in autophagy.However,its function and molecular mechanism in tumor cell migration are still un-known.The role of STX5 in influencing hepatocellular carci-noma(HCC)is an important topic in our research.Methods:By using quantitative reverse transcription polymerase chain reaction(qPCR),western blotting,and immunohistochemical analysis of RNA and protein in tissues,we comprehensively evaluated data sets from public databases and clinical patient cohorts for STX5.The correlation of STX5 expression with the clinicopathological characteristics of HCC patients were assessed.In addition,we predicted signal pathways from dif-ferentially expressed genes(DEGs)and the Cancer Genome Atlas(TCGA)databases,and confirmed the prediction using integrated transcriptome and RNA-seq.We further investi-gated the underlying mechanisms of STX5 in the migration and adhesion of HCC cells both in vitro and in vivo.Results:In the TCGA dataset and our patient cohort,STX5 levels were significantly higher in HCC tissues than in adjacent normal liver tissues.At the same time,high expression of STX5 pre-dicted worse prognosis in patients with liver cancer.High ex-pression of STX5 indicates the decrease of adhesion and the increase of migration of HCC cells,and the conversion of epi-thelial-mesenchymal transition(EMT)in vitro via PI3K/mTOR pathway activation.Conversely,when Sirolimus,a phospho-inositide 3-kinase(PI3K)/AKT/mechanistic target of rapa-mycin(mTOR)inhibitor acts on cells simultaneously,STX5 overexpression-mediated enhancement of HCC metastasis is reversed.Double-negative regulation of STX5 and mTOR further enhanced the inhibitory effect of STX5 on HCC me-tastasis.In vivo,STX5 knockdown inhibited the metastasis of HCC cells.Conclusions:Our study demonstrates a novel research result that STX5 promotes HCC metastasis through PI3K/mTOR pathway.We believe that combined inhibition of STX5 and mTOR is a potential treatment for effectively pro-longing patient survival and inhibiting HCC metastasis.

Engineering blood-brain barrier-permeable and tumor cell-ingestible pro-proteins for glioblastoma treatment

Intracellular protein therapeutics holds great potentials for the treatment of glioblastoma, which however, is greatly challenged by the unmet demands to concomitantly penetrate the blood-brain barrier(BBB) and glioblastoma cell membrane barrier with high efficiency and selectivity. Herein, a unique pro-protein platform was developed via facile green synthesis, which allowed efficient and selective delivery into glioblastoma cells in a carrier-free manner. Pro-proteins were engineered via reversible modification of native proteins in the aqueous buffer with 3,4-dihydroxy-phenylalanine, the substrate of L-type amino acid transporter(LAT1), bridged with a phenylboronic acid-containing linker. By harnessing the LAT1-mediated direct transport mechanism, the optimized pro-protein, named protein-M2-D, can efficiently penetrate BBB after i.v. injection, and subsequently enable selective and endocytosis-free delivery of various proteins including enzymes, toxins, and antibodies into glioblastoma cells, wherein intracellular H_(2)O_(2) triggered traceless restoration of the native protein structure. Systemic administration of saporin-M2-D provoked potent anti-tumor efficacy against orthotopic U87 glioblastoma in mice, without inducing systemic toxicity. Such a facile, versatile, and robust platform renders a promising paradigm for cytosolic protein delivery and glioblastoma treatment.

iRGD-reinforced, photo-transformable nanoclusters toward cooperative enhancement of intratumoral penetration and antitumor efficacy

Insufficient intratumoral penetration greatly hurdles the anticancer performance of nanomedicine. To realize highly efficient tumor penetration in a precisely and spatiotemporally controlled manner, far-red light-responsive nanoclusters (NCs) capable of size shrinkage and charge conversion were developed and co-administered with iRGD to synergistically improve the intratumoral penetration and the anticancer efficacy. The NCs were constructed using the singlet oxygen-sensitive (SOS) polyethylene glycolpolyurethane-polyethylene glycol (PEG-(1O2)PU-PEG) triblock copolymer to encapsulate the doxorubicin (DOX)-loaded, chlorin e6 (Ce6)-conjugated polyamindoamine (PAMAM) dendrimer (DCD) via the double-emulsion method. Co-administration of iRGD notably increased the permeability of NCs within tumor vasculature and tumor tissues. In addition, upon far-red light irradiation (660 nm) of tumors at low optical density (10 mW/cm2), the generated 1O2 could disintegrate the NCs and release the DCD with positive surface charge and ultra-small size (~ 5 nm), which synergized with iRGD to enable deep intratumoral penetration. Consequently, the local 1O2 at lethal concentrations along with the released DOX efficiently and cooperatively eradicated tumor cells. This study provides a convenient approach to spatiotemporally promote the intratumoral penetration of nanomedicine and mediate programmed anticancer therapy.

Cardiomyocyte-targeted anti-inflammatory nanotherapeutics against myocardial ischemia reperfusion (IR) injury

Myocardial ischemia reperfusion(IR)injury is closely related to the overwhelming inflammation in the myocardium.Herein,cardiomyocyte-targeted nanotherapeutics were developed for the reactive oxygen species(ROS)-ultrasensitive co-delivery of dexamethasone(Dex)and RAGE small interfering RNA(siRAGE)to attenuate myocardial inflammation.PPTP,a ROSdegradable polycation based on PGE2-modified,PEGylated,ditellurium-crosslinked polyethylenimine(PEI)was developed to surface-decorate the Dex-encapsulated mesoporous silica nanoparticles(MSNs),which simultaneously condensed siRAGE and gated the MSNs to prevent the Dex pre-leakage.Upon intravenous injection to IR-injured rats,the nanotherapeutics could be efficiently transported into the inflamed cardiomyocytes via PGE2-assisted recognition of over-expressed E-series of prostaglandin(EP)receptors on the cell membranes.Intracellularly,the over-produced ROS degraded PPTP into small segments,promoting the release of siRAGE and Dex to mediate effective RAGE silencing(72%)and cooperative antiinflammatory effect.As a consequence,the nanotherapeutics notably suppressed the myocardial fibrosis and apoptosis,ultimately recovering the systolic function.Therefore,the current nanotherapeutics represent an effective example for the codelivery and on-demand release of nucleic acid and chemodrug payloads,and might find promising utilities toward the synergistic management of myocardial inflammation.

Macrophage-targeting gene silencing orchestrates myocardial microenvironment remodeling toward the anti-inflammatory treatment of ischemia-reperfusion (IR) injury

Ischemia-reperfusion (IR) injury represents a major cause of myocardial dysfunction after infarction and thrombolytic therapy, and it is closely related to the free radical explosion and overwhelming inflammatory responses. Herein, macrophage-targeting nanocomplexes (NCs) are developed to mediate efficient co-delivery of siRNA against MOF (siMOF) and microRNA-21 (miR21) into myocardial macrophages, cooperatively orches-trating the myocardial microenvironment against IR injury. Bioreducible, branched poly(β-amino ester) (BPAE-SS) is designed to co-condense siMOF and miR21 into NCs in a multivalency-reinforced approach, and they are surface-decorated with carboxylated mannan (Man-COOH) to shield the positive surface charges and enhance the serum stability. The final MBSsm NCs are efficiently internalized by myocardial macrophages after systemic administration, wherein BPAE-SS is degraded into small segments by intracellular glutathione to promote the siMOF/miR21 release, finally provoking efficient gene silencing. Thus, cardiomyocyte protection and macro-phage modulation are realized via the combined effects of ROS scavenging, inflammation inhibition, and autophagy attenuation, which ameliorates the myocardial microenvironment and restores the cardiac function via positive cellular crosstalk. This study renders promising solutions to address the multiple systemic barriers against in vivo nucleic acid delivery, and it also offers new options for IR injury by manipulating multiple reciprocal bio-reactions.