Biomedical Applications of Single Protein Nanoparticles

The drug carrier function of single protein nanoparticles, i.e., each individual protein molecule covered by a very thin, porous and few nanometer thick polymer layer, has been investigated. This layer around protein molecule is very thin, about 3-5 nm thick and highly porous, thus it does not reduce seriously the enzymatic function of protein molecule. The spatial structure of encapsulated protein molecule, which is essential in its function, can be stabilized by this polymer layer. Bovine serum albumin was used as protein drug molecule and it was encapsulated with acrylamide-bisarylamide random copolymer. The polymerization, starting from the modified sites of the surface of bovine serum albumin molecules was initiated by TEMED （tetramethylethylenediamine）. These single albumin nanoparticles were painted with fluorescein isothiocyanate. This material was then injected into the inferior vena cava of rats. The treated rats were decapitated after 1 to 10 minutes and its brain was investigated by fluorescent microscopy. It was proved that bovine serum albumin molecules as drugs encapsulated in polymer nano-layer with a reduced size （about 10 nm） can pass through the blood brain barrier. The results suggest that this method is capable of transformation of biomacromolecules to access the brain tissue via the blood.

Payload-free protein nanoparticles target inflamed colons to restore intestinal barrier integrity for effectively treating inflammatory bowel diseases

Anti-inflammatory compounds,delivered as a payload to the gastrointestinal tract(GIT)by carriers,still cannot treat inflammatory bowel diseases without avoid-ing side effects.Here,we developed payload-free protein nanoparticles(PNPs)that crossed GIT to retain in the colon and treat colitis by restoring intestinal bar-rier integrity by modulating gut microbiome and metabolome.Specifically,PNPs,orally administered to mice with acute colitis,reached the colon within three hours.Consequently,PNPs improve gut microbiota dysbiosis to reverse metabolism bal-ance,suppressing the expression of tumor-necrosis factorαand toll-like receptor 4 that restores the intestinal barrier integrity.PNPs then ameliorated colon inflam-mation and attenuated gut microbiota dysbiosis by exerting probiotic effects on gut microbiota,treating colitis in a week more effectively than the clinically often used 5-aminosalicylic acid without causing undesired side effects.Such PNPs repre-sent safe,sustainable,and cost-effective therapeutics for treating inflammatory and metabolic diseases by eliminating microbial and metabolomic imbalance.

Targeted delivery of a STING agonist to brain tumors using bioengineered protein nanoparticles for enhanced immunotherapy

Immunotherapy is emerging as a powerful tool for combating many human diseases.However,the application of this life-saving treatment in serious brain diseases,including glioma,is greatly restricted.The major obstacle is the lack of effective technologies for transporting therapeutic agents across the blood-brain barrier(BBB)and achieving targeted delivery to specific cells once across the BBB.Ferritin,an iron storage protein,traverses the BBB via receptor-mediated transcytosis by binding to transferrin receptor 1(TfR1)overexpressed on BBB endothelial cells.Here,we developed bioengineered ferritin nanoparticles as drug delivery carriers that enable the targeted delivery of a small-molecule immunomodulator to achieve enhanced immunotherapeutic efficacy in an orthotopic glioma-bearing mouse model.We fused different glioma-targeting moieties on self-assembled ferritin nanoparticles via genetic engineering,and RGE fusion protein nanoparticles(RGE-HFn NPs)were identified as the best candidate.Furthermore,RGE-HFn NPs encapsulating a stimulator of interferon genes(STING)agonist(SR717@RGE-HFn NPs)maintained stable self-assembled structure and targeting properties even after traversing the BBB.In the glioma-bearing mouse model,SR717@RGE-HFn NPs elicited a potent local innate immune response in the tumor microenvironment,resulting in significant tumor growth inhibition and prolonged survival.Overall,this biomimetic brain delivery platform offers new opportunities to overcome the BBB and provides a promising approach for brain drug delivery and immunotherapy in patients with glioma.

Fe_3O_4@PAM@NTA-Ni^(2+) Magnetic Composite Nanoparticles for Highly Specific Separation of His-tagged Proteins

A facile approach has been developed to synthesize Fe3O4@PAM（polyacrylamide） nanoparticles（NPs） with carboxyl groups on the surfaces by copolymerization with acrylamide and acrylic acid in Fe3O4 NPs aqueous suspension. Nitrilotriacetic acid（NTA） was conjugated to the magnetic NPs via well-known carboniimide chemistry using EDC and NHS. The Ni^（2＋） ions loaded on the surface of NPs provide abundant docking sites for immobilization of His-tagged green fluorescent proteins（His-tagged GFP）. The high magnetic property of Fe3O4@PAM@NTA-Ni^（2＋） allows an easy separation of the NPs from solution under an external magnetic field, with high His-tagged protein binding capacity（42 μg protein/mg of NPs）. The NPs can be recycled for at least four times without significant loss of binding capacity to proteins. These materials show great potential to separate His-tagged protein with low-cost purification at industrial scale.

Completely Green Synthesis of Ag Nanoparticles Stabilized by Soy Protein Isolate under UV Irradiation

A completely green pathway for the preparation of Ag nanoparticles was proposed, by using soy protein isolate （SPI） as stabilizer under UV irradiation and H2O as the environmentally benign solvent throughout the preparation. Transmission electronic microscopy （TEM） and zeta potential characterization results indicated that the Ag nanoparticles were stable and well dispersed with an average diameter about 13 nm, and X-ray diffraction （XRD） analysis of SPI/Ag composite nanoparticles confirmed the formation of metallic silver. UV-Vis spectrum showed that the Ag nanoparticles dispersion solution had the maximum absorbance at about 430 nm due to surface plasmon resonance of the Ag nanoparticles. Infrared spectroscopy confirmed that the polypeptide backbone of SPI was not cleaved during the conjugation process and that some active amino groups were oxidized. The SPI/Ag composite nanoparticles have excellent antibacterial activity against two representative bacteria, staphylococcus aureus （Gram positive） and escherichia coli （Gram negative） in the presence of SPI.

Toward innovative veterinary nanoparticle vaccines

Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SAPNs),which have shown potential for enhancing antigen-presenting cell uptake,B-cell activation,and lymph node trafficking.Numerous nanovaccines have been utilized in veterinary medicine,including natural self-assembled protein nanoparticles,rationally designed self-assembled protein nanoparticles,animal virus-derived nanoparticles,bacteriophagederived nanoparticles,and plant-derived nanoparticles,which will be discussed in this review.SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases.This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine.Furthermore,this paper highlights their disadvantages and methods for improving their immunogenicity.

Self-assembling protein nanocarrier for selective delivery of cytotoxic polypeptides to CXCR4^(+) head and neck squamous cell carcinoma tumors

Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma(HNSCC) mortality. The overexpression of chemokine receptor 4(CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4^(+)tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4^(+)HNSCC cells, achieving a high accumulation in CXCR4^(+)tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4^(+)cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted proteinonly nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.

mRNA delivery in cancer immunotherapy

Messenger RNA(mRNA)has drawn much attention in the medical field.Through various treatment approaches including protein replacement therapies,gene editing,and cell engineering,mRNA is becoming a potential therapeutic strategy for cancers.However,delivery of mRNA into targeted organs and cells can be challenging due to the unstable nature of its naked form and the low cellular uptake.Therefore,in addition to mRNA modification,efforts have been devoted to developing nanoparticles for mRNA delivery.In this review,we introduce four categories of nanoparticle platform systems:lipid,polymer,lipid-polymer hybrid,and protein/peptide-mediated nanoparticles,together with their roles in facilitating mRNA-based cancer immunotherapies.We also highlight promising treatment regimens and their clinical translation.