Molecular Mechanisms of Intracellular Delivery of Nanoparticles Monitored by an Enzyme‑Induced Proximity Labeling

Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and subcellular targets,it is essential to explore the delivery of nanomedicines at the molecular level.However,due to the lack of technical methods,the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date.Here,we develop an enzyme-induced proximity labeling technology in nanoparticles(nano-EPL)for the real-time monitoring of proteins that interact with intracellular nanomedicines.Poly(lactic-co-glycolic acid)nanoparticles coupled with horseradish peroxidase(HRP)were fabricated as a model(HRP(+)-PNPs)to evaluate the molecular mechanism of nano delivery in macrophages.By adding the labeling probe biotin-phenol and the catalytic substrate H_(2)O_(2)at different time points in cellular delivery,nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles.Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP(+)-PNPs in macrophages over time.Based on dynamic clustering analysis of these proteins,we further discovered that different organelles,including endosomes,lysosomes,the endoplasmic reticulum,and the Golgi apparatus,are involved in delivery with distinct participation timelines.More importantly,the engagement of these organelles differentially affects the drug delivery efficiency,reflecting the spatial–temporal heterogeneity of nano delivery in cells.In summary,these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.

Micro and Nanotechnology for Intracellular Delivery Therapy Protein

Proteins therapy is of great importance in the treatment of protein deficiency disease. Most human diseases are related to the malfunctioning of one or more proteins. The most effective and direct approach is protein therapy, which delivers the proteins into the target cell to replace the dysfunction protein and maintain the balance of organism. However, clinical application is frequently hampered by various biological barriers to their successful delivery. This review aims to discuss the recent advances about microparticles and nanoparticles fabricated using micro and nanotechnology for intracellular delivery therapy protein and give some suggestions about the promising delivery system.

Recent electroporation-based systems for intracellular molecule delivery

Intracellular delivery of functional molecules,such as DNA probes and plasmids,is an important method for investigating cellular mechanisms and changing cell fates in biomedicine.Among various delivery methods,recent years have seen the emergence of electroporation-based techniques that provide versatile platforms for molecule delivery,with high efficiency and controlled dosage.In this Review,we describe recent electroporation-based systems for intracellular molecule delivery.The principles of electroporation for cell membrane perforation and cargo delivery are briefly summarized.Focusing on various scenarios for the application of electroporation,we review electroporation devices that variously employ structures based on nanochannels,nanostraws,and flow-through microfluidic channels for in vitro intracellular molecule delivery.We also consider in vivo targeted therapies based on delivery of active molecules by electroporation according to the lesion locations.Finally,we discuss the current challenges facing electroporation-based techniques,as well as opportunities for their future development,which may lead to innovations in intracellular molecule delivery both for cellular analysis in the laboratory and treatment in the clinic.

Targeted and intracellular delivery of protein therapeutics by a boronated polymer for the treatment of bone tumors

The treatment of malignant bone tumors by chemotherapeutics often receives poor therapeutic response due to the specific physiological bone environment,and thus calls for the development of new therapeutic options.Here,we reported a bone-targeted protein nanomedicine for this purpose.Saporin,a toxin protein,was co-assembled with a boronated polymer for intracellular protein delivery,and the formed nanoparticles were further coated with an anionic polymer poly(aspartic acid)to shield the positive charges on nanoparticles and provide the bone targeting function.The prepared ternary complex nanoparticles showed high bone accumulation both in vitro and in vivo,and could reverse the surface charge property from negative to positive after locating at tumor site triggered by tumor extracellular acidity.The boronated polymer in the de-shielded nanoparticles further promote intracellular delivery of saporin into tumor cells,exerting the anticancer activity of saporin by inactivation of ribosomes.As a result,the bone-targeted and saporin-loaded nanomedicine could kill cancer cells at a low saporin dose,and efficiently prevented the progression of osteosarcoma xenograft tumors and bone metastatic breast cancer in vivo.This study provides a facile and promising strategy to develop protein-based nanomedicines for the treatment of malignant bone tumors.

Protein@PP-Zn nanocomplex assembled by coordination of zinc ions used for intracellular protein delivery

In recent years,intracellular delivery of protein drugs has attracted great attention,and polymer-based systems have been extensively exploited to develop efficient and safe carriers.However,efficient intracellular delivery of protein drugs remains a challenge because of the cell membrane barrier and endosome entrapment.Herein,we report a protein@PP-Zn nanocomplex,which consists of an imidazole-containing block polymer poly(ethylene glycol)-block-poly(β-amino ester)(PEG-b-PAE(Im),PP),zinc ions,and protein drugs,for efficient intracellular protein delivery.PEG-b-PAE(Im)could conjugate proteins via the bridging effect of zinc ions which simultaneously coordinate with imidazole groups on polymer and electron donor groups,such as imidazole and primary amine groups,on protein to improve the loading stability of proteins.Under a slightly acidic environment near cancer cells,the protonation of PAE(Im)backbone increases the positive charge density of the nanocomplex and promotes endocytosis.While under a more acidic environment in endosomes,further protonation of imidazole groups leads to the disintegration of the nanocomplex and the breakdown of endosomes because of the proton sponge effect.Finally,protein is released into the cytoplasm.With the assistance of the nanocomplex,proteins with different sizes and isoelectric points are effectively delivered into cells.This work provides a stable,efficient and universal strategy for intracellular protein delivery.

Use of stimulatory responsive soft nanoparticles for intracellular drug delivery

Drug delivery has made tremendous advances in the last decade.Targeted therapies are increasingly common,with intracellular delivery highly impactful and sought after.Intracellular drug delivery systems have limitations due to imprecise and non-targeted release profiles.One way this can be addressed is through using stimuli-responsive soft nanoparticles,which contain materials with an organic backbone such as lipids and polymers.The choice of biomaterial is essential for soft nanoparticles to be responsive to internal or external stimuli.The nanoparticle must retain its integrity and payload in non-targeted physiological conditions while responding to particular intracellular environments where payload release is desired.Multiple internal and external factors could stimulate the intracellular release of drugs from nanoparticles.Internal stimuli include pH,oxidation,enzymes,while external stimuli include ultrasound,light,electricity,magnetic fields.Stimulatory responsive soft nanoparticulate systems specifically utilized to modulate intracellular delivery of drugs are explored in this review.

Combinatorial synthesis of redox-responsive cationic polypeptoids for intracellular protein delivery application

Biologics play an essential role in treating various indications from cancers to the metabolic diseases,while the current development of new classes of intracellular-acting protein drugs is still hindered because of high molecular mass and overall hydrophilicity of proteins creating extremely poor permeability across cell membrane.Hence,there remains an unmet need to develop safe,potent approaches to augment intracellular protein delivery efficiency.Here,we described a facile multicomponent reaction system for generating a small library of redox-responsive cationic polypeptoids with high biocompatibility.The co-assembly of optimized polymer with protein leads to the formation of compacted nanocomplexes with smaller size and high encapsulation efficiency,thus improving cellular internalization via the macropinocytosis and/or caveolae-mediated endocytosis mainly.After endo-lysosomal escape,the nanocomplexes can be disassociated to efficiently release cargo proteins into the cytosol,owing to the intracellular glutathione(GSH)-triggered rapid cleavage of disulfide bonds in polymers backbone.As a result,we screened a promising platform reagent for efficient cytosolic protein delivery application.

A green light-enhanced cytosolic protein delivery platform based on BODIPY-protein interactions

Development of cytosolic protein delivery platforms brings new possibilities for various incurable diseases.Strategies based on polymer/protein self-assembly have shown their potential in protein delivery.However,versatile photocontrolled platforms based on self-assembly for protein delivery are seldom reported.Herein,we report a boron-dipyrromethene(BODIPY)-modified polyamidoamine(PAMAM)with excellent photo-controllability and efficiency for the cytosolic delivery of various proteins.High serum stability was achieved by coating hyaluronic acid and human serum albumin on the surface of BODIPY-modified PAMAM/protein nanoparticles.The nanoparticles under green light irradiation allowed efficient intracellular delivery of multiple cargo proteins with different charges and molecular weights and promoted endosome escape.The study provides valuable guidance for the development of BODIPY derivative-based protein delivery systems and advances the research in intracellular protein delivery.

Targeted pH-responsive polyion complex micelle for controlled intracellular drug delivery

Cancer the rapy with nanoscale drug formulations has made significant progress in the past few decades.However,the selective accumulation and release of therapeutic agents in the lesion sites are still great challenges.To this end,we developed a cRGD-decorated pH-responsive polyion complex(PIC)micelle for intracellular targeted delivery of doxorubicin(DOX)to upregulate tumor inhibition and reduce toxicity.The PIC micelle was self-assembled via the electrostatic interaction between the positively charged cRGD-modified poly(ethylene glycol)-block-poly(L-lysine)and the anionic acid-sensitive 2,3-dimethylmaleic anhydride-modified doxorubicin(DAD).The decoration of cRGD enhanced the cell internalization of PIC micelle through the specific recognition ofαvβ3 integrin on the membrane of tumor cells.The active DOX was released under intracellular acidic microenvironment after endocytosis following the decomposition of DAD.Moreover,the targeted PIC micelle exhibited enhanced inhibition efficacies toward hepatoma in vitro and in vivo compared with the insensitive controls.The smart multifunctional micelle provides a promising platform for target intracellular delivery of therapeutic agent in cancer therapy.

Boronic acid-rich dendrimer for efficient intracellular peptide delivery

Interests in intracellular peptide delivery have continued to grow,significantly fueled by the importance of peptides and their mimetics in modern cell biology and pharmaceutical industry.However,efficient intracellular delivery of membrane-impermeable peptides of different polarities remains a challenging task.In this study,we develop a general and robust strategy for intracellular peptide delivery by using a boronic acid-rich dendrimer.The designed material is capable of transporting peptides with different polarities and charge properties into the cytosol of various cell lines without inducing additional cytotoxicity.The transduction efficacy and proteolytic stability of cargo peptides delivered by the boronic acid-rich dendrimer are much superior to peptides conjugated with cell penetrant peptides such as octaarginine.In addition,the bioactivities of pro-apoptotic peptides are maintained after intracellular delivery.This study provides a versatile and robust platform for the intracellular delivery of membrane-impermeable peptides.

Virus-inspired nanoparticles as versatile antibacterial carriers for antibiotic delivery against Gram-negative and Gram-positive bacteria

Infectious diseases become one of the leading causes of human death. Traditional treatment based on classical antibiotics could not provide enough antibacterial activity to combat bacterial infections due to low bioavailability, even leading to antibiotic resistance. In recent years, biomimetic delivery systems have been developed to improve drug therapy for various diseases, such as malignant tumor and cardiovascular disease. In this work, we designed virus-inspired nanodrugs(VNDs) through co-assembly of amphiphilic lipopeptide dendrons and poly(lactic-co-glycolic acid) polymers for high-efficiency antibiotic delivery. These VNDs had well-defined and stable nanostructures for tetracycline encapsulation and delivery. The VNDs were capable of promoting antibiotic internalization and enhancing their antibacterial effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Additionally, no obvious cytotoxicity of VNDs was observed to human cell lines. This work successfully demonstrated the virus-mimetic nanoparticles served as promising and applicable antibiotic delivery platform for antibacterial treatment.

A homologous and molecular dual-targeted biomimetic nanocarrier for EGFR-related non-small cell lung cancer therapy

The abnormal activation of epidermal growth factor receptor(EGFR)drives the development of non-small cell lung cancer(NSCLC).The EGFR-targeting tyrosine kinase inhibitor osimertinib is frequently used to clinically treat NSCLC and exhibits marked efficacy in patients with NSCLC who have an EGFR mutation.However,free osimertinib administration exhibits an inadequate response in vivo,with only~3%patients demonstrating a complete clinical response.Consequently,we designed a biomimetic nanoparticle(CMNP^(@Osi))comprising a polymeric nanoparticle core and tumor cell-derived membrane-coated shell that combines membrane-mediated homologous and molecular targeting for targeted drug delivery,thereby supporting a dual-target strategy for enhancing osimertinib efficacy.After intravenous injection,CMNP^(@Osi)accumulates at tumor sites and displays enhanced uptake into cancer cells based on homologous targeting.Osimertinib is subsequently released into the cytoplasm,where it suppresses the phosphorylation of upstream EGFR and the downstream AKT signaling pathway and inhibits the proliferation of NSCLC cells.Thus,this dual-targeting strategy using a biomimetic nanocarrier can enhance molecular-targeted drug delivery and improve clinical efficacy.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation

Fluorescence microscopy is the method of choice for studying intracellular dynamics.However,its success depends on the.availability of specific and stable markers.A prominent example of markers that are rapidly gaining interest are nanobodies(Nbs.-15 kDa),which can be functionalized with bright and photostable organic fluorophores.Due to their relatively small size and high specificity,Nbs offer great potential for high-quality long-term subcellular imaging,but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells.We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability.Being a laser-based technology,it is readily compatible with light microscopy and the typical cell recipients used for that.Spurred by these promising initial results,we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells.We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm,actin-bundling protein Fascin,and the histone H2A/H2B heterodimers.With an efficiency of more than 80%labeled cells and minimal toxicity(-2%),photoporation proved to be an excellent intracellular delivery method for Nbs.Time-lapse microscopy revealed that cell division rate and migration remained unaffected,confirming excellent cell viability and functionality.We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells,laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

Synthesis of Protein Nano-Conjugates for Cancer Therapy

A eukaryotic cell contains thousands of proteins that regulate its cellular function; delivering functional proteins into cells to rectify cellular functions holds great promise for treatment of various diseases, especially cancers. In this context, ribonuclease （RNase）, an enzyme that breaks down ribonucleic acid （RNA）, has great potential for cancer therapy. However, its therapeutic application is hampered by poor intracellular delivery efficiency and inhibition by ubiquitous intracellular RNase inhibitors. In this work, by designing and synthesizing RNase nano-conjugates by in situ atom transfer radical polymerization （ATRP）, we demonstrate a simple solution to address both challenges. Compared with native RNase, nano-conjugates exhibit significantly enhanced intracellular delivery efficiency, inhibitor resistance, and a near five-fold increase in cytotoxicity. This work provides a novel platform for delivery of therapeutic proteins for cancer therapy and other applications.

Photothermal scaffolds/surfaces for regulation of cell behaviors

Regulation of cell behaviors and even cell fates is of great significance in diverse biomedical applications such as cancer treatment,cell-based therapy,and tissue engineering.During the past decades,diverse methods have been developed to regulate cell behaviors such as applying external stimuli,delivering exogenous molecules into cell interior and changing the physicochemical properties of the substrates where cells adhere.Photothermal scaffolds/surfaces refer to a kind of materials embedded or coated with photothermal agents that can absorb light with proper wavelength(usually in near infrared region)and convert light energy to heat;the generated heat shows great potential for regulation of cell behaviors in different ways.In the current review,we summarize the recent research progress,especially over the past decade,of using photothermal scaffolds/surfaces to regulate cell behaviors,which could be further categorized into three types:(i)killing the tumor cells via hyperthermia or thermal ablation,(ii)engineering cells by intracellular delivery of exogenous molecules via photothermal poration of cell membranes,and(iii)releasing a single cell or an intact cell sheet via modulation of surface physicochemical properties in response to heat.In the end,challenges and perspectives in these areas are commented.