Self-assembled Protein Nanoparticles Research Articles

Development of recombinant protein-based nanoparticle systems for inducing tumor cell apoptosis: In vitro evaluation of their cytotoxic and apoptotic effects on cancer cells

Myeloid leukemia cell differentiation protein (Mcl-1) is a B-cell lymphoma 2 (Bcl-2) family antiapoptotic protein that regulates cellular apoptosis. Mcl-1 expression is important for cancer cell survival and a prospective target in cancer treatment. Bcl-2 homology 3 (BH3) mimetic peptides, such as the Mcl-1-specific MS1 peptide that targets conserved BH3 domains, are highly specific Mcl-1 inhibitors. However, the application of these peptides is limited due to their metabolic instability, degradation by cellular proteases, and low cell permeability. In this study, it was aimed to form nanostructures to increase the cellular uptake and stability of the Mcl-1 inhibitor MS1 peptide. The Bim BH3 mimetic MS1 peptide was produced recombinantly in fusion with mNeonGreen fluorescent protein and polyhistidine (6xHisTag). Self-assembled protein nanoparticles were fabricated from the recombinant MS1-mNeonGreen protein. Furthermore, the MS1-mNeonGreen protein was encapsulated into polymeric nanoparticles. The cytotoxic effects of these nanoparticles on cancer cells [cervical cancer cell line (HeLa), lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231)] and healthy cell line [Human umbilical vein endothelial cells (HUVEC)] was analyzed by MTT test. The apoptotic effect of nanoparticles on these cells was investigated by determining the antiapoptotic and proapoptotic protein levels. As a result, it was revealed that the nanoparticles have a specific cytotoxic effect against the cells and induce apoptosis.

Utilization of self-assembled soy protein nanoparticles as carriers for natural pigments: Examining non-interaction mechanisms and stability

In this study, we made self-assembled soy protein nanoparticles (SPN) by partially hydrolyzing soy protein isolate (SPI) using Flavourzyme at different hydrolysis times (0, 0.5, 1, 1.5, 2, 3, and 4 h). Compared with SPI, SPN (hydrolysis time, 1.5 h) was spherical with smaller particle size, better dispersion, and higher surface hydrophobicity. The interaction and stability of SPN loaded with different concentrations (0, 0.05, 0.1, 0.3, and 0.5 mmol/L) of lutein (Lut), β-carotene (β-Car), and curcumin (Cur) were then characterized. The encapsulation efficiency, load capacity, and X-ray diffraction measurements showed that natural pigments were successfully encapsulated by SPN in amorphous form, with the highest encapsulation efficiency observed at 0.05 mmol/L with 94.3%, 88.7%, and 96.0% for Lut, β-Car, and Cur, respectively. Fluorescence, ultraviolet, and infrared spectroscopy revealed that natural pigments were loaded into SPN mainly by hydrophobic interactions, and hydrogen bonding was also involved in the loading process of Lut and Cur. Moreover, the outcomes of the isothermal titration calorimetry measurement validated the spontaneous and entropy-driven binding process between SPN and natural pigments. Due to its low molecular weight, a high number of phenolic hydroxyl groups, and low spatial resistance, Cur was most encapsulated in SPN, while the strongest interactions were observed. Lut, β-Car, and Cur were encapsulated by SPN with significantly better thermal and light stability, especially Cur. This study may open a new avenue for the use of SPN as product formulations to extend lipophilic natural edible pigments in food products.

Enhanced curcumin transportation across epithelial barrier by mucus-permeable soy protein nanoparticles-mediated dual transcytosis pathways

Nanocarrier-delivered bioactive compounds are highly desirable for their improved stability and applicability, but their bioavailability is still limited due to the strong mucus and epithelial cell barriers. Herein, a series of self-assembled soy protein nanoparticles (SPNPs) with different mucus permeabilities were prepared and their delivery efficiency upon Curcumin (Cur) encapsulation was evaluated. Results demonstrated that the formed SPNPs-Cur exhibited high compatibility and cellular antioxidant accessibility. Besides, SPNPs enhanced the cellular uptake and transmembrane permeation of Cur, especially promoted the transportation of proto-Cur in addition to Cur metabolites. The SPNPs with the rapid mucus diffusion capacity presented more efficient transcytosis across the Caco-2 cell monolayer, which was mediated by a combination of paracellular and transcellular pathways. This work verified that mucus-permeable soy protein nanoparticles could be a promising delivery system for improving the bioavailability of bioactive compounds.

Engineered Norovirus-Derived Nanoparticles as a Plug-and-Play Cancer Vaccine Platform.

In recent years, virus-derived self-assembled protein nanoparticles (NPs) have emerged as attractive antigen delivery platforms for developing both preventive and therapeutic vaccines. In this study, we exploited the genetically engineered Norovirus S domain (Nov-S) with SpyCatcher003 fused to the C-terminus to develop a robust, modular, and versatile NP-based carrier platform (Nov-S-Catcher003). The NPs can be conveniently armed in a plug-and-play pattern with SpyTag003-linked antigens. Nov-S-Catcher003 was efficiently expressed in Escherichia coli and self-assembled into highly uniform NPs with a purified protein yield of 97.8 mg/L. The NPs presented high stability at different maintained temperatures and after undergoing differing numbers of freeze-thaw cycles. Tumor vaccine candidates were easily obtained by modifying Nov-S-Catcher003 NPs with SpyTag003-linked tumor antigens. Nov-S-Catcher003-antigen NPs significantly promoted the maturation of bone marrow-derived dendritic cells in vitro and were capable of efficiently migrating to lymph nodes in vivo. In TC-1 and B16F10 tumor-bearing mice, the subcutaneous immunization of NPs elicited robust tumor-specific T-cell immunity, reshaped the tumor microenvironment, and inhibited tumor growth. In the TC-1 model, the NPs even completely abolished established tumors. In conclusion, the Nov-S-Catcher003 system is a promising delivery platform for facilitating the development of NP-based cancer vaccines.

Structural and functional properties of self-assembled peanut protein nanoparticles prepared by ultrasonic treatment: Effects of ultrasound intensity and protein concentration

Peanut protein isolate (PPI) nanoparticles were prepared by self-assembly under the combined action of ultrasound (US) and protein concentration. The effects of ultrasound intensity (150–500 W) and protein concentration (1–12 %, w/v) on the structural and functional properties of PPI nanoparticles were investigated. Low-intensity US significantly increased the particle size of PPI, but high-intensity US decreased it. The largest PPI nanoparticles were obtained when 10 % PPI was subjected to low-intensity US treatment (200 W for 5 min). These nanoparticles possessed unique structural characteristics, such as the lowest absolute ζ-potential and the highest contents of exposed free sulfhydryl and disulfide bond, which may be responsible for their excellent heat-set gelling properties. The 12 % PPI treated with low- and high-intensity US had the highest emulsifying activity index and emulsifying stability index, respectively. The self-assembled PPI nanoparticles induced by US treatments at high protein concentrations have great potentials for application in the food industry.

Modified Stability of microRNA-Loaded Nanoparticles.

microRNAs represent promising drugs to treat and prevent several diseases, such as diabetes mellitus. microRNA delivery brings many obstacles to overcome, and one strategy to bypass them is the manufacturing of self-assembled microRNA protein nanoparticles. In this work, a microRNA was combined with the cell-penetrating peptide protamine, forming so-called proticles. Previous studies demonstrated a lack of microRNA dissociation from proticles. Therefore, the goal of this study was to show the success of functionalizing binary proticles with citric acid in order to reduce the binding strength between the microRNA and protamine and further enable sufficient dissociation. Thus, we outline the importance of the present protons provided by the acid in influencing colloidal stability, achieving a constant particle size, and monodispersing the particle size distribution. The use of citric acid also provoked an increase in drug loading. Against all expectations, the AFM investigations demonstrated that our nanoparticles were loose complexes mainly consisting of water, and the addition of citric acid led to a change in shape. Moreover, a successful reduction in binding affinity and nanoparticulate stability are highlighted. Low cellular toxicity and a constant cellular uptake are demonstrated, and as uptake routes, active and passive pathways are discussed.

Avidin-Biotin functionalized self-assembled protein nanoparticles as EGFR targeted therapeutics for the treatment of lung cancer: characterization and cell viability

Gefitinib targeting efficiency can be modified via enhancing its hydrophobicity and poor bioavailability by conjugating it with biotin-streptavidin conjugated Bovine serum albumin nanoparticles as a hotspot in the field of biological therapy for human cancer. The study aims to develop and characterize lyophilized Biotin-Streptavidin conjugated Bovine serum albumin nanoparticles containing Gefitinib prepared by desolvation method. As protein being the most amendable to surface functionalization were prepared by conjugating them with strongest interaction, that is, Biotin-Streptavidin. The Desolvation method is based on nanoprecipitation method that reduces the solubility of drug and polymer in aqueous solution by using desolvating agents in order to prepare protein nanoparticles that were further functionalized by biotinylation followed with streptavidin conjugation for enhancement in affinity and specificity. Protein NPs were found to be monodisperse with particle size in range of 130 ± 5.131 and 174 ± 3.055 with the approximate negatively surface charge zeta potential of −9 mv. The drug loading and the entrapment efficiency was found to be 16% and 74 ± 2.64% with 8 days of sustained drug release at the physiological pH. Cytotoxicity studies after 72 h shown better cell growth inhibition with biotinylated streptavidin conjugated bovine serum albumin nanoparticles containing gefitinib with reduced side effect and more apoptosis of cells as compared to gefitinib loaded bovine serum albumin nanoparticles and free gefitinib.

Self-assembled soy protein nanoparticles by partial enzymatic hydrolysis for pH-Driven Encapsulation and Delivery of Hydrophobic Cargo Curcumin

Controlled partial enzymatic hydrolysis has been shown to be an efficacious strategy for devising and construction of multifunctional soy protein nanoparticles (SPNPs). In this work, we reported a successful fabrication of novel SPNPs obtained from self-assembly of the amphiphilic hydrolysate after partial hydrolysis of soy protein isolate (DH, 4%), and explored their potential as a nanocarrier for active cargo delivery. Curcumin was effectively loaded into the hydrophobic core of SPNPs by utilizing the pH-driven method, and the resultant curcumin-loaded SPNPs were spherical with a small particle size (80 nm in diameter), homogeneous size distribution and relatively high encapsulation efficiency (78%). The solubility and stability of curcumin against different NaCl concentrations (0–300 mM) and temperatures (75–95 °C) were remarkably enhanced by encapsulation into SPNPs. Additionally, SPNPs were able to effectively protect curcumin from degradation or precipitation during simulated gastric-intestinal digestion, showing a significantly enhanced bioaccessibility. Encapsulated curcumin was valid in alleviating cell oxidative damage induced by H2O2, mainly by scavenging intracellular free radicals, inhibiting lipid oxidation, and elevating endogenous antioxidant enzymes levels. Interestingly, the vehicle SPNPs showed synergistic antioxidant effect, creatively evidencing their bifunctionality in cellular antioxidant activity.

Mechanism of enhancing the water-solubility and stability of curcumin by using self-assembled cod protein nanoparticles at an alkaline pH

Curcumin (Cur) is a bioactive phytochemical which is claimed to have several health-promoting benefits, whose applications are challenging due to its poor water-solubility, chemical instability, and low bioavailability. In this research, Cur was encapsulated in the cod protein (CP) using a pH-driven method to enhance its solubility and stability. The physicochemical and structural properties of cod protein-curcumin nanoparticles (CP-Cur) formed were characterized. Fluorescence spectroscopy (FL), ultraviolet spectroscopy (UV), circular dichroism (CD), and dynamic light scattering (DLS) results collectively suggest that the protein originally with a molten-globule state refolded into a more ordered structure after neutralization, during which Cur was incorporated. Fluorescence quenching and isothermal titration calorimetry (ITC) further showed that the CP/Cur binding was mainly driven by hydrophobic interactions, resulting in static fluorescence quenching and energy release. Up to 99.50% of Cur was loaded in the CP delivery system. Furthermore, the thermal stability and photostability of Cur were greatly improved due to the protection of the protein. The present study proved that cod protein could be a great potential edible carrier for encapsulating curcumin.

Design of integrin αvβ3 targeting self-assembled protein nanoparticles with RGD peptide

Integrin αvβ3 was reported as positive regulators of tumorigenesis and highly expressed in cancer stem cells and kinds of cancers, thus, it is an appealing target for cancer treatment. Nanomedicine with targeting delivery ability has developed rapidly and shown its great therapeutic potential in cancer therapy. Proteins are ideal material for nanomedicine regarding to their excellent biocompatibility, and protein-only self-assembled nanoparticles technology provides a robust method to produce protein nanoparticles. Pro-apoptotic proteins or peptides, such as BAK, have attracted increasing attention in the inhibition of tumor growth. However, the self-assembled nanoparticles of BAK targeting to integrin αvβ3 over-expressed tumor cells need to be investigated. In this study, we designed recombinant proteins with BH3 BAK as active domain and RGD peptides as targeting ligands to self-assemble into protein nanoparticles (named as PN2-1 et al.), then experimentally evaluated the nanoparticle size, fluorescence feature, stability, targeting ability and cytotoxicity to tumor cells in vitro. The results showed that the protein nanoparticles containing RGD peptides had a uniform particle size with an diameter of approximately 23 nm. PN2-1 had notable inhibition to cell proliferation of C6 cells, C26 cells and MCF-7 cells, with a lower IC50 than the nanoparticles which only had BAK motif without RGD peptide. PN2-1 had higher cellular uptake into C6 cells than MCF-7 cells. Our results demonstrate that the RGD peptide could enhance the cytotoxicity of BAK nanoparticles to tumor cells and increase their tumor targeting ability. This study provides an insight into the design and development of integrin αvβ3 targeting protein nanoparticle for cancer treatment.

Targeted delivery of polypeptide nanoparticle for treatment of traumatic brain injury.

Background and purpose: Traumatic brain injury (TBI) is a major disease without effective treatment. Recently, Tat-NR2B9c peptide emerged as a promising neuroprotective agent, but limited in clinical translation by it low brain penetrability. We synthesized Tat-NR2B9c loaded self-assembled activatable protein nanoparticles, termed TN-APNPs, and demonstrated that TN-APNPs enhanced the delivery of Tat-NR2B9c to the brain lesion in stroke. Herein we developed a novel approach to further engineering TN-APNPs for targeted delivery of Tat-NR2B9c to the injured brain with enhanced efficiency through conjugation of CAQK or CCAQK, a short peptide.Methods: Short peptide-conjugated TN-APNPs were synthesized by conjugated with CAQK or CCAQK via a click condensation reaction with CBT, then analyzed by dynamic light scattering, transmission electron microscopy and thrombin responsive assay. Characterization of short peptide-conjugated TN-APNPs were investigated by using cell excitotoxicity assay and transwell blood-brain-barrier model in vitro, and pharmacokinetics, IVIS imaging system and confocal analysis in TBI-bearing mice. Evaluation of therapeutic effects were analyzed by H&E staining, Elevated Plus Maze analysis and Rotarod test.Results: CAQK-conjugated TN-APNPs (C-TN-APNPs) and CCAQK-conjugated TN-APNPs (CC-TN-APNPs) were spherical in morphology and 30 nm in diameter. In vitro studies revealed that TN-APNPs, C-TN-APNPs and CC-TN-APNPs were responsive to thrombin cleavage, reduced the cytotoxicity of Tat-NR2B9c, and increased BBB permeability of Tat-NR2B9c. CC-TN-APNPs demonstrated the better circulation time, better targeting ability and penetrating efficiency to the injured brain, and better therapeutic benefits in vivo studies.Conclusion: This study demonstrated CC-TN-APNPs as a promising therapeutic for clinical management of TBI.

A Novel Self-Assembled Mitochondria-Targeting Protein Nanoparticle Acting as Theranostic Platform for Cancer.

Self-assembled protein nanoparticles have attracted much attention in biomedicine because of their biocompatibility and biodegradability. Protein nanoparticles have become widely utilized as diagnostic or therapeutic agents for various cancers. However, there are no reports that protein nanoparticles can specifically target mitochondria. This targeting is desirable, since mitochondria are critical in the development of cancer cells. In this study, the discovery of a novel self-assembled metal protein nanoparticle, designated GST-MT-3, is reported, which targets the mitochondria of cancer cells within 30 min in vitro and rapidly accumulates in tumors within 1 h in vivo. The nanoparticles chelate cobalt ions [GST-MT-3(Co2+ )], which induces reactive oxygen species (ROS) production and reduces the mitochondrial membrane potential. These effects lead to antitumor activity in vivo. GST-MT-3(Co2+ ) with covalently conjugated paclitaxel synergistically suppress tumors and prolong survival. Importantly, the effective dosage of paclitaxel is 50-fold lower than that utilized in standard chemotherapy (0.2 vs 10 mg kg-1 ). To the best of the authors' knowledge, GST-MT-3 is the first reported protein nanoparticle that targets mitochondria. It has the potential to be an excellent platform for combination therapies.

Fabrication of self-assembled Radix Pseudostellariae protein nanoparticles and the entrapment of curcumin

Simulating the process of traditional Chinese medicine decoction, Radix Pseudostellariae protein (RPP)-based nanoparticles were constructed by combining heat treatment with pH adjustment in succession. The formed nanoparticles were characterized as homogeneously dispersed sphere within 100 nm in diameter. With curcumin as a drug model, the potential application of RPP as a nanocarrier was studied. Curcumin could combine to RPP through hydrophobic interaction and quench the intrinsic fluorescence of RPP. Results of X-ray diffraction revealed that the crystal formation of curcumin was suppressed after the formation of nanocomplexes. In addition, the curcumin-loaded nanocomplexes exhibited good thermal stability and the light stability of curcumin was significantly improved. The curcumin-loaded nanocomplexes had stronger reducing power than free curcumin, which displayed additive effect between curcumin and RPP. In summary, the obtained RPP nanoparticles are potential to become new drug delivery carriers in food field and pharmaceutical applications for the encapsulation of hydrophobic components.

Genetically engineered and self-assembled oncolytic protein nanoparticles for targeted cancer therapy.

The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E.coli, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E.coli. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment.

Encapsulation of Aconitine in Self-Assembled Licorice Protein Nanoparticles Reduces the Toxicity In Vivo.

Many herbal medicines and compositions are clinically effective but challenged by its safety risks, i.e., aconitine (AC) from aconite species. The combined use of Radix glycyrrhizae (licorice) with Radix aconite L. effectively eliminates toxicity of the later while increasing efficacy. In this study, a boiling-stable 31-kDa protein (namely GP) was purified from licorice and self-assembled into nanoparticles (206.2 ± 2.0 nm) at pH 5.0, 25 °C. The aconitine-encapsulated GP nanoparticles (238.2 ± 1.2 nm) were prepared following the same procedure and tested for its toxicity by intraperitoneal injection on ICR mouse (n = 8). Injection of GP-AC nanoparticles and the mixed licorice-aconite decoction, respectively, caused mild recoverable toxic effects and no death, while the aconitine, particle-free GP-AC mixture and aconite decoction induced sever toxic effects and 100 % death. Encapsulation of poisonous alkaloids into self-assembled herbal protein nanoparticles contributes to toxicity attenuation of combined use of herbs, implying a prototype nanostructure and a universal principle for the safer clinical applications of herbal medicines.

Formulating tumor-homing peptides as regular nanoparticles enhances receptor-mediated cell penetrability

Homing peptides are exploited in nanomedicine to functionalize either free drugs or nanostructured materials used as drug carriers. However, the influence of multivalent versus monovalent peptide presentation on the interaction with the receptor and on the consequent intracellular delivery of the associated cargo remains poorly explored. By using a tumor-homing peptide (T22) with regulatable self-assembling properties we have investigated here if its display in a either a monomeric form or as multimeric, self-assembled protein nanoparticles might determine the efficacy of receptor-mediated penetrability into target cells. This has been monitored by using a fluorescent cargo protein (iRFP), which when fused to the homing peptide acts as convenient reporter. The results indicate that the nanoparticulate protein versions are significantly more efficient in mediating receptor-dependent uptake than their unassembled counterparts. These finding stresses an additional benefit of nanostructured materials based on repetitive building blocks, regarding the multivalent presentation of cell ligands that facilitate cell penetration in drug delivery applications.