Release Model of Water-soluble Chitosan Nanoparticles for Protein Delivery

[Objective] The experiment aimed to explore release rule of water-soluble chitosan (WSC) in vitro. [Method]The bovine serum albumin(BSA) was taken as a model protein drug and some existing release models such as Kinetics model, Gompertz model, Weibull model, Higuchi model and Logistic model were used to fit the BSA release profile from WSC carriers. [Result] Except Higuchi model and Logistic model, other models could fit BSA release profile better. [Conclusion] Gompertz two-order kinetics model could fit the release of WSC nano-particles better and model parameters had practical physical meaning.

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.

Traceless photodegradable polymer cocoons for universal protein delivery and light-controlled gene editing

Polymer conjugation was found highly valuable in clinic to improve the bioavailability of protein therapeutics.However,it is still a tremendous challenge to achieve a complete release of original proteins from the conjugated hybrid under external stimulus to recover active proteins in the targeted tissue.Herein,we report a general light-controlled protein delivery methodology by weaving a photodegradable polymer cocoon around proteins,which could reliably protect them from degradation in the dark while efficiently releasing them under illumination without any residual atoms.The surface charge of the polymer shell is easily tunable to facilitate efficient cell uptake.The versatility of this strategy is demonstrated by the delivery of the Cas9/sg RNA complex that realized light-controlled gene editing both in vitro and in vivo,and such repertoire is of particular value in regard to minimizing the off-target toxicity of CRISPR-Cas9-based gene therapy.

Lipocoacervate,a tunable vesicle for protein delivery

Controlled delivery of proteins and other biologics is a growing medium of therapy for diseases previously untreatable.Here we report a self-assembling,tunable vesicle for the controlled delivery of growth factors and cytokines.Coacervate made of heparin and a biocompatible polycation,PEAD,forms the core of the vesicle;lipids form the membrane of the vesicle.We call this vesicle lipocoacervate(LipCo),which has a high affinity for growth factors and cytokines due to heparin.LipCo is a tunable protein delivery vehicle.The vesicle size is controlled through polymer and salt concentrations.Membrane functionalization enables potential for targeting capabilities with long-term storage through lyophilization.Importantly,the controlled delivery of therapeutics also avoids high toxicity to treated cells in vitro.Here we report on these key principles of LipCo assembly and design.

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.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Polymers have been widely proposed as carriers for cytosolic protein delivery despite multiple barriers such as protein binding,cell internalization,and endosome escape during cytosolic delivery.Inspired by the strong binding affinity of natural polyphenols with proteins and cell membranes,herein we propose polyphenol modification to improve the efficacy of the protein delivery of cationic polymers.Catecholmodified dendrimers with balanced hydrophobic and hydrogen-bonding interactions show the highest efficacy for various cargo proteins and peptides while the pyrogallol-grafted ones exhibit the lowest efficacy due to increased ligand hydrophilicity.The catechol-based polymers efficiently deliver various bioactive proteins into the cytosol of live cells,exerting biofunctions after intracellular release,and successfully transmittingα-chymotrypsin into tumor cells in vivo to inhibit tumor growth.This study proves that polycatechols can serve as a family of highly efficient carriers for delivery of macromolecular biopharmaceuticals.

Microfluidic one-step, aqueous synthesis of size-tunable zeolitic imidazolate framework-8 for protein delivery

Zeolitic imidazolate framework-8(ZIF-8)with porous structure,biocompatibility,and pH-sensitive release behavior is a promising nanoplatform for protein delivery.However,it is still a challenging task for a practical synthesis of protein-loaded ZIF-8 nanoparticles.Here we report an all-aqueous microfluidic reactor for one-step,rapid,and highly controlled synthesis of ZIF-8 nanoparticles with high protein loading at room temperature.Microfluidic reactor allows for an ultrafast(<35 ms),complete mixing of Zn2+ions and 2-methylimidazole(2-MIM)at different molecular ratios,leading to the formation of stable ZIF-8 nanoparticles with tunable sizes(13.2–191.4 nm)in less than 30 s.By pre-mixing various proteins such as bovine serum albumin(BSA)(isoelectric point(pI)=5.82),ovalbumin(OVA)(pI=4.82),or RNase A(pI=8.93)with 2-MIM,ZIF-8 nanoparticles can be synthesized with protein encapsulation efficiency over 97%.Among the nanoparticles with different sizes,25 nm ZIF-8 nanoparticles show the best performance in promoting the cellular uptake of protein payload.Using OVA as a model protein,we demonstrate that 25 nm ZIF-8 nanoparticles significantly enhance the cytosolic delivery of antigen,as indicated by the effective activation of dendritic cells.We anticipate that this microfluidic synthesis of nanomaterials may advance the emerging field of cytosolic protein delivery.

Tailoring Cationic Helical Polypeptides for Efficient Cytosolic Protein Delivery

Protein delivery is of central importance for both diagnostic and therapeutic applications.However,protein delivery faces challenges including poor endosomal escape and thus limited efficiency.Here,we report the facile construction and screening of a small library of cationic helical polypeptides for cytosolic protein delivery.The library is based on a random copolymer poly(γ-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}esteryl-L-glutamate)-randompoly(γ-6-chlorohexyl-L-glutamate)[P(EG3-r-ClC6)Glu],which is then modified with various pyridine derivatives and alkyl thiols.Flow Cytometry,confocal laser scanning microscopy,and viability assay collaboratively identify two leading polymers,showing efficient delivery of enhanced green fluorescent protein(eGFP)and low cytoto-xicity.This finding is further validated by the cytosolic delivery of RNase A and cytochrome C(Cyt C)to HeLa cells in the viability assay.Together,this work demonstrates that high-throughput screening is an effective and viable approach to the selection of cationic helical polypeptides for the cytosolic delivery of functional proteins.

Study the lipidoid nanoparticle mediated genome editing protein delivery using 3D intestinal tissue model

Lipid nanoparticles are promising carriers for oral drug delivery.For bioactive cargos with intracellular targets,e.g.gene-editing proteins,it is essential for the cargo and carrier to remain complexed after crossing the epithelial layer of intestine in order for the delivery system to transport the cargos inside targeted cells.However,limited studies have been conducted to verify the integrity of cargo/carrier nanocomplexes and their capability in facilitating cargo delivery intracellularly after the nanocomplex crossing the epithelial barrier.Herein,we used a traditional 2D transwell system and a recently developed 3D tissue engineered intestine model and demonstrated the synthetic lipid nanoparticle(carrier)and protein(cargo)nanocomplexes are able to cross the epithelial layer and deliver the protein cargo inside the underneath cells.We found that the EC16-63 LNP efficiently encapsulated the GFP-Cre recombinase,penetrated the intestinal monolayer cells in both the 2D cell culture and 3D tissue models through temporarily interrupting the tight junctions between epithelial layer.After transporting across the intestinal epithelia,the EC16-63 and GFP-Cre recombinase nanocomplexes can enter the underneath cells to induce gene recombination.These results suggest that the in vitro 3D intestinal tissue model is useful for identifying effective lipid nanoparticles for potential oral drug delivery.

An Intracellular pH-Actuated Polymer for Robust Cytosolic Protein Delivery

Robust cytosolic protein delivery requires both efficient protein binding with delivery vehicles and effective protein release after cell internalization.Although a variety of stimuli-responsive carriers have been designed,simultaneously integrating these two functions in one versatile carrier is challenging.Herein,we developed a polyamidoamine(PAMAM)-based polymer with an intracellular pHactuated hydrophobic-to-hydrophilic transition for this purpose.

Design and Preparation of pH-responsive Curdlan Hydrogels as a Novel Protein Delivery Vector

New pH-responsive saccharide hydrogels were designed and prepared using curdlan derivatives（curdlan-Bochistidine, CUR-HIS）. The CUR-HIS hydrogels possessed highly porous structures. The swelling ratios of CUR-HIS hydrogels increased with the degree of substitution of Boc-histidine groups. And the addition of 0.5 mol/L Na Cl provoked a sharp reduction of swelling ratio of CUR-HIS hydrogels. Bovine serum albumin（BSA） can be efficiently encapsulated into CUR-HIS hydrogels. Moreover, the release profiles of BSA at different p H values from CUR-HIS hydrogels were significantly different. These hydrogels showed good biocompatibility in the cytotoxicity assays. The CUR-HIS hydrogels are of great potential in biomedical applications such as protein delivery systems.

Enzyme-instructed hybrid nanogel/nanofiber oligopeptide hydrogel for localized protein delivery

Enzyme-catalysis self-assembled oligopeptide hydrogel holds great interest in drug delivery,which has merits of biocompatibility,biodegradability and mild gelation conditions.However,its application for protein delivery is greatly limited by inevitable degradation of enzyme on the encapsulated proteins leading to loss of protein activity.Moreover,for the intracellularly acted proteins,cell membrane as a primary barrier hinders the transmembrane delivery of proteins.The internalized proteins also suffer from acidic and enzymatic degradation in endosomes and lysosomes.We herein develop a proteasemanipulated hybrid nanogel/nanofiber hydrogel for localized delivery of intracellularly acted proteins.The embedded polymeric nanogels(CytoC/aNGs)preserve activity of cytochrome c(CytoC)that is an intracellular activator for cell apoptosis as a model protein against proteolysis,and do not affect the gelation properties of the protease-catalysis assembled hydrogels.The injectable hydrogel(CytoC/aNGs/Gel)serves as a reservoir to enhance intratumoral retention and realize sustainable release of CytoC/aNGs.The released CytoC/aNGs increase cellular uptake of CytoC and enhance its intracellular delivery to its target site,cytoplasm,resulting in favorable apoptosis-inducing and cytotoxic effects.We show that a single local administration of CytoC/aNGs/Gel efficiently inhibit the tumor growth in the breast tumor mouse model.

Engineered extracellular vesicles enable high-efficient delivery of intracellular therapeutic proteins

Developing an intracellular delivery system is of key importance in the expansion of protein-based therapeutics acting on cytosolic or nuclear targets.Recently,extracellular vesicles(EVs)have been exploited as next-generation delivery modalities due to their natural role in intercellular communication and biocompatibility.However,fusion of protein of interest to a scaffold represents a widely used strategy for cargo enrichment in EVs,which could compromise the stability and functionality of cargo.Herein,we report intracellular delivery via Ev-based approach(IDEA)that efficiently packages and delivers native proteins both in vitro and in vivo without the use of a scaffold.As a proof-of-concept,we applied the IDEA to deliver cyclic GMP-AMP synthase(cGAS),an innate immune sensor.The results showed that cGAS-carrying EVs activated interferon signaling and elicited enhanced antitumor immunity in multiple syngeneic tumor models.Combining cGAS EVs with immune checkpoint inhibition further synergistically boosted antitumor efficacy in vivo.Mechanistically,scRNA-seq demonstrated that cGAS EVs mediated significant remodeling of intratumoral microenvironment,revealing a pivotal role of infiltrating neutrophils in the antitumor immune milieu.Collectively,IDEA,as a universal and facile strategy,can be applied to expand and advance the developmentof protein-based therapeutics.

PLGA-based implants for sustained delivery of peptides/proteins:Current status,challenge and perspectives

Peptide and protein drugs with therapeutic effects suffer from their short half-life and low stability,albeit their high efficiency and specificity.To overcome these demerits,long-acting drug delivery systems have been developed,wherein poly(lactic-co-glycolic acid)(PLGA)implants are most preferred owing to their excellent biodegradability and biocompatibility.Dozens of PLGA based products have been approved since1986,when the first product,named Decapeptyl R,successfully marched into market.To meet the increasing demand for delivering various peptides and proteins,different kinds of technologies have been developed for lab-scale fabrication or industrial manufacture.This review aims to introduce recent advances of PLGA implants,and give a brief summary of fundamental properties of PLGA,fabrication technologies of peptides/proteins-loaded PLGA implants as well as factors influencing the drug release processes.Moreover,challenges and future perspectives are also highlighted.

An orally administered bacterial membrane protein nanodrug ameliorates doxorubicin cardiotoxicity through alleviating impaired intestinal barrier

The cardiotoxicity caused by Dox chemotherapy represents a significant limitation to its clinical application and is a major cause of late death in patients undergoing chemotherapy.Currently,there are no effective treatments available.Our analysis of 295 clinical samples from 132 chemotherapy patients and 163 individuals undergoing physical examination revealed a strong positive correlation between intestinal barrier injury and the development of cardiotoxicity in chemotherapy patients.We developed a novel orally available and intestinal targeting protein nanodrug by assembling membrane protein Amuc_1100(obtained from intestinal bacteria Akkermansia muciniphila),fluorinated polyetherimide,and hyaluronic acid.The protein nanodrug demonstrated favorable stability against hydrolysis compared with free Amuc_1100.The in vivo results demonstrated that the protein nanodrug can alleviate Dox-induced cardiac toxicity by improving gut microbiota,increasing the proportion of short-chain fatty acid-producing bacteria from the Lachnospiraceae family,and further enhancing the levels of butyrate and pentanoic acids,ultimately regulating the homeostasis repair of lymphocytes in the spleen and heart.Therefore,we believe that the integrity of the intestinal barrier plays an important role in the development of chemotherapy-induced cardiotoxicity.Protective interventions targeting the intestinal barrier may hold promise as a general clinical treatment regimen for reducing Dox-induced cardiotoxicity.

Designing the new generation of intelligent biocompatible carriers for protein and peptide delivery

Therapeutic proteins and peptides have revolutionized treatment for a number of diseases, and the expected increase in macromolecule-based therapies brings a new set of challenges for the pharmaceutics field. Due to their poor stability, large molecular weight, and poor transport properties,therapeutic proteins and peptides are predominantly limited to parenteral administration. The short serum half-lives typically require frequent injections to maintain an effective dose, and patient compliance is a growing issue as therapeutic protein treatments become more widely available. A number of studies have underscored the relationship of subcutaneous injections with patient non-adherence, estimating that over half of insulin-dependent adults intentionally skip injections. The development of oral formulations has the potential to address some issues associated with non-adherence including the interference with daily activities, embarrassment, and injection pain. Oral delivery can also help to eliminate the adverse effects and scar tissue buildup associated with repeated injections. However, there are several major challenges associated with oral delivery of proteins and peptides, such as the instability in the gastrointestinal(GI)tract, low permeability, and a narrow absorption window in the intestine. This review provides a detailed overview of the oral delivery route and associated challenges. Recent advances in formulation and drugdelivery technologies to enhance bioavailability are discussed, including the co-administration of compounds to alter conditions in the GI tract, the modification of the macromolecule physicochemical properties, and the use of improved targeted and controlled release carriers.

Tailoring combinatorial lipid nanoparticles for intracellular delivery of nucleic acids, proteins,and drugs

Lipid nanoparticle(LNP)-based drug delivery systems have become the most clinically advanced non-viral delivery technology.LNPs can encapsulate and deliver a wide variety of bioactive agents,including the small molecule drugs,proteins and peptides,and nucleic acids.However,as the physicochemical properties of small-and macromolecular cargos can vary drastically,every LNP carrier system needs to be carefully tailored in order to deliver the cargo molecules in a safe and efficient manner.Our group applied the combinatorial library synthesis approach and in vitro and in vivo screening strategy for the development of LNP delivery systems for drug delivery.In this Review,we highlight our recent progress in the design,synthesis,characterization,evaluation,and optimization of combinatorial LNPs with novel structures and properties for the delivery of small-and macromolecular therapeutics both in vitro and in vivo.These delivery systems have enormous potentials for cancer therapy,antimicrobial applications,gene silencing,genome editing,and more.We also discuss the key challenges to the mechanistic study and clinical translation of new LNP-enabled therapeutics.

Design of Polymers for Intracellular Protein and Peptide Delivery

Cytosolic protein delivery techniques are of great importance for cell biology,biotechnology and protein drug development.The design of carriers with robust efficiency in cytosolic protein delivery is challenging.This account provides a progress report of polymeric carriers for this purpose in our group.During the past years,we have developed several types of functionalized polymers for cytosolic protein and peptide delivery by engineering polymers with ligands such as guanidinium,boronate,coordination ligands and fluoroalkyls.The designed polymers showed improved protein/peptide binding affinities,and successfully delivered various cargo proteins into the cytosol of living cells,while maintaining their bioactivity.In addition,the polymers showed potent efficiencies in the delivery of tumor antigens,therapeutic peptides,toxins and antioxidant proteins in vivo.We hope these polymers could be translated for protein delivery in the treatment of various diseases in the future.

Sol-gel synthesis,properties and protein loading/delivery capacity of hollow bioactive glass nanospheres with large hollow cavity and mesoporous shell

Hollow nanospheres exhibit unique properties and find a wide interest in several potential applications such as drug delivery.Herein,novel hollow bioactive glass nanospheres(HBGn)with large hollow cavity and large mesopores in their outer shells were synthesized by a simple and facile one-pot ultrasound assisted sol-gel method using PEG as the core soft-template.Interestingly,the produced HBGn exhibited large hollow cavity with ~43 nm in diameter and mesoporous shell of ~37 nm in thickness and 7 nm pore size along with nanosphere size around 117 nm.XPS confirmed the presence of Si and Ca elements at the surface of the HBGn outer shell.Notably,HBGn showed high protein loading capacity(~570 mg of Cyto c per 1 g of HBGn)in addition to controlled protein release over 5 d.HBGn also demonstrated a good in vitro capability of releasing calcium(Ca^(2+):170 ppm)and silicate(SiO_(4)^(4-):78 ppm)ions in an aqueous medium over 2 weeks under physiological-like conditions.Excellent in vitro growth of bone-like hydroxyapatite nanocrystals was exhibited by HBGn during the soaking in SBF.A possible underlying mechanism involving the formation of spherical aggregates(coils)of PEG was proposed for the formation process of HBGn.

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.