Protein-based Drug Delivery Systems Research Articles

An insight into the effect of interaction with protein on antibacterial activity of chitosan derivatives

Antimicrobial activity of chitosan in protein-rich media is of a particular interest for various protein-based drug delivery and other systems. For the first time, bacteriostatic activity of chitosan derivatives in the presence of caseinate sodium (CAS) was studied and discussed. Complexation of chitosan derivatives soluble in acidic (CH and RCH) or alkalescent (RCH) media with CAS was confirmed by fluorescent spectroscopy, turbodimetry, light scattering data and measurement of electrical potentials of CAS/chitosan derivative complexes. An addition of CH and RCH caused a static quenching of CAS. Binding constants Kb determined for CH/CAS and RCH/CAS complexes at pH 6.0 were equal to 29.8 × 106 M−1 and 8.9 × 106 M−1, respectively. Kb value of RCH/CAS complex at pH 7.4 was equal to 1.1 × 105′M−1. The poisoned food method was used for counting the number and the direct measurement of the size of bacterial colonies on the surfaces of turbid agar media containing CAS/chitosan derivative complexex. Complete suppression of E. coli cells growth and restriction of S. aureus cells growth were observed on the surface of acidic media. A high concentration of CAS reduced the activity. The activity of RCH in alkalescent media is low or absent. These results can be promising for preparation of microbiologically stable protein-based drug delivery systems.

Degradation Kinetic Studies of BSA@ZIF-8 Nanoparticles with Various Zinc Precursors, Metal-to-Ligand Ratios, and pH Conditions.

Nanosized zeolitic imidazolate framework particles (ZIF-8 nanoparticles [NPs]) have strong potential as effective carriers for both in vivo and in vitro protein drug delivery. Synthesis of ZIF-8 and stability of protein encapsulation within ZIF-8 are affected by several factors, notably the metal ion source and molar ratio. To systematically investigate these factors, we investigated such effects using BSA as a model test protein to synthesize BSA@ZIF-8 NPs at various metal-to-ligand (M:L) ratios. SEM, FTIR, XRD, and DLS were applied to characterize the morphology and structure of BSA@ZIF-8 NPs and their effects on protein loading capacity. Degradation kinetics and protein release behavior of BSA@ZIF-8 NPs were evaluated at pH 5.0 (to simulate the tumor environment) and pH 7.4 (to mimic the blood environment). Our objective was to define optimal combinations of the high protein loading rate and rapid release under varying pH conditions, and we found that (i) the yield of BSA@ZIF-8 NPs decreased as the M:L ratio increased, but the protein content increased. This highlights the need to strike a balance between cost-effectiveness and practicality when selecting ZIF-8 NPs with different molar ratios for protein-based drug formulation. (ii) BSA@ZIF-8 NPs exhibited cruciate flower-like shapes when synthesized using Zn(NO3)2 as the zinc precursor at M:L ratios of 1:16 or 1:20. In all other cases, the NPs displayed a regular rhombic dodecahedral structure. Notably, the release behavior of the NPs did not differ significantly between these morphologies. (iii) When Zn(OAc)2 was used as the zinc precursor, the synthesized ZIF-8 NPs exhibited a smaller size compared to the Zn(NO3)2-derived ZIF-8 NPs. (iv) The release rate and amount of BSA protein were higher at pH 5.0 compared to pH 7.4. (v) Among the different formulations, BSA@ZIF-8 with an M:L ratio of 1:16 at pH 5.0 was observed to have a shorter time to reach a plateau (0.5 h) and higher protein release, making it suitable for locally rapid administration in a tumor environment. BSA@ZIF-8 prepared from Zn(OAc)2 at an M:L ratio of 1:140 showed the slower release of BSA protein over a 24-h period, indicating its suitability for sustained release delivery. In conclusion, our findings provide a useful basis for the practical application of ZIF-8 NPs in protein-based drug delivery systems.

Synthesis and Evaluation of BSA-Loaded PLGA-Chitosan Composite Nanoparticles for the Protein-Based Drug Delivery System.

The purpose of this study was to synthesize composite nanoparticles (NPs) based on poly(d,l-lactic-co-glycolic acid) (PLGA) and chitosan (CS) and evaluate their suitability for the delivery of protein-based therapeutic molecules. Composite NPs possess a unique property which is not exhibited by any other polymer. Unlike other polymers, only the composite NPs lead to improved transfection efficiency and sustained release of protein. The composite NP were prepared by grafting CS on the surface of PLGA NPs through EDC-NHS coupling reaction. The size of bovine serum albumin (BSA)-loaded PLGA NPs and BSA-loaded PLGA-CS composite NPs was 288 ± 3 and 363 ± 4 nm, respectively. The zeta potential of PLGA NPs is -18 ± 0.23, and that of composite particles is 19 ± 0.40, thus confirming the successful addition of CS on the surface of PLGA NPs. Composite NPs were characterized using dynamic light scattering, scanning/transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, release profile, and gel electrophoresis. The encapsulation efficiency of PLGA NPs was 88%. These composite NPs were easily uptaken by the A549 cell line with no or minimal cytotoxicity. The present study emphasizes that the composite NPs are suitable for delivery of BSA into the cells with no cytotoxicity or very little cytotoxicity, while maintaining the integrity of the encapsulated BSA.

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin.

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

Bioengineered Human Serum Albumin Fusion Protein as Target/Enzyme/pH Three-Stage Propulsive Drug Vehicle for Tumor Therapy.

Human serum albumin (HSA) has the characteristics of biocompatibility and long circulation, which is widely used as the carrier of insoluble anticancer drugs, but it also has some disadvantages such as weak tumor targeting and uncontrollable drug release. Herein, HSA was modified to improve its biological performance by introducing polyhistidine (pHis), matrix metalloproteinase-2 (MMP-2) digestion, and Arg-Gly-Asp (RGD) peptide at the separated end of HSA through gene fusion technology. The resulting protein expressed by Pichia pastoris could self-assemble into 3RGD-HSA-MMP-18His nanoparticles (RHMH18 NPs) accompanied by loading hydrophobic drug paclitaxel (PTX) into the polyhistidine micelle core. RHMH18 NPs exhibited active tumor targeting in high efficiency owing to the RGD-mediated specific binding toward ανβ3-integrin upregulated on tumor vasculature endothelium, resulting in the enrichment of therapeutic substances in tumor sites. Once reaching the tumor microenvironment, RHMH18 NPs was cut off by MMP-2 to remove the HSA-3RGD moiety, leaving the small and positively charged histidine micelle, which could penetrate the deep part of tumor tissue more effectively. Finally, the histidine micelle escaped from lysosome successfully and released drug in response to pH. The in vivo experiments' results demonstrated that the three-stage propulsion RHMH18 NPs presented superior tumor inhibition activity with minimal side effects, providing potential strategies of protein based drug delivery systems for tumor therapy.

Multifunctionalized Protein-Based Drug Delivery System for Inhibition of Tumor Growth and Progression.

To improve the bioavailability of hydrophobic drugs and realize tumor targeting therapeutic actions efficiently, a nanosized multifunctional protein-based drug delivery system was constructed by self-assembly in a facile manner. Negatively charged cRGD-conjugated bovine serum albumin (cRGD-BSA) loaded with a hydrophobic antitumor drug (curcumin, CUR) was complexed with electropositive protamine sulfate (PS) via electroattractive forces to form CUR@cRGD-BSA/PS nanoparticles. Flow cytometry and confocal microscopy show that the multifunctional CUR@cRGD-BSA/PS nanoparticles lead to significantly increased intracellular drug accumulation in tumor cells owing to the tumor specific affinity of cRGD ligands as well as the membrane translocating property of PS. As a result, the multifunctional protein-based delivery system (CUR@cRGD-BSA/PS) exhibits an apparently enhanced inhibitory efficiency on malignant cells as compared with free CUR and the monofunctional delivery system (CUR@cRGD-BSA). More importantly, the expression of proteins (Bcl-2, cyclin D1, β-catenin, c-Myc, and MMP-9) involved in cancer development in the tumor cells treated by CUR@cRGD-BSA/PS is dramatically downregulated, implying the functional protein-based drug delivery system can effectively prevent tumor progression. Our investigation gives insight into the construction of multifunctional protein-based delivery carriers for tumor targeting delivery of hydrophobic drugs.

Comparative evaluation for controlling release of niacin from protein- and cellulose-chitosan based hydrogels

This work deals with assessing the efficient performance of sodium caseinate (SC) as protein-based drug delivery system of niacin (NA) than carboxymethyl cellulose (CMC). In this respect the hydrogels from complexation of chitosan with sodium caseinate (SC/Ch) or sodium carboxymethyl cellulose (CMC/Ch) were prepared. The Synthesized NA free and loaded hydrogels were characterized by many techniques for examining the interaction, morphology, swelling, encapsulation efficiency (EE) and loading (L) % of niacin, as well as cytotoxicity study. The finding data showed the promising behavior of SC/Ch hydrogel than CMC/Ch hydrogel, toward the amount of loaded NA (95.6%) and in vitro slow sustained release up to 24 h. Whereas, the entrapment efficiency of the CMC/Ch to nicotinic acid was reached 85.6%, and it possessed highly initial burst release followed by a slower release up to 24 h. At pH 7.4 (simulated intestinal fluid) both hydrogels provided higher level of releasing profile to NA than pH 2.1 (gastric fluid). The NA release from hydrogels followed Fickian and non-Fickian diffusion mechanism according to pH 7.4 and 2.1, respectively. It is interesting to note that, the data obtained are higher than those obtained from literature reported hydrogel, e.g., poly (2-hydroxyethyl methacrylate). Neutral red uptake and lactate dehydrogenase assays confirmed both hydrogels have good biocompatibility and could be used as nontoxic drug delivery system. So, we recommended SC/Ch hydrogel as an effective controlled niacin drug delivery system with reducing systemic side effects and improved intestinal targeting efficiency.

Bioengineered elastin- and silk-biomaterials for drug and gene delivery.

Advances in medical science have led to diverse new therapeutic modalities, as well as enhanced understanding of the progression of various disease states. These findings facilitate the design and development of more customized and exquisite drug delivery systems that aim to improve therapeutic indices of drugs to treat a variety of conditions. Synthetic polymer-based drug carriers have often been the focus of such research. However, these structures suffer from challenges with heterogeneity of the starting material, limited chemical features, complex functionalization methods, and in some cases a lack of biocompatibility. Consequently, protein-based polymers have garnered much attention in recent years due to their monodisperse features, ease of production and functionalization, and biocompatibility. Genetic engineering techniques enable the advancement of protein-based drug delivery systems with finely tuned physicochemical properties, and thus an expanded level of customization unavailable with synthetic polymers. Of these genetically engineered proteins, elastin-like proteins (ELP), silk-like proteins (SLP), and silk-elastin-like proteins (SELP) provide a unique set of alternatives for designing drug delivery systems due to their inherent chemical and physical properties and ease of engineering afforded by recombinant DNA technologies. In this review we examine the advantages of genetically engineered drug delivery systems with emphasis on ELP and SLP constructions. Methods for fabrication and relevant biomedical applications will also be discussed.

Exploiting iron-binding proteins for drug delivery.

Currently, many therapies fail due to an insufficient drug dose reaching the target site and high systemic toxicity. Protein-based drug delivery systems that allow an increase in drug concentration at a specific location in the body or predominantly target malignant cells are promising technologies. Due to the high need for iron in many disorders including various types of cancer, iron-binding proteins: transferrin, ferritin and hemoglobin, are a promising tool as drug carriers. In this review we summarize the characteristics of human iron-binding proteins and present their use in targeted drug delivery strategies.

Encapsulation and Systemic Delivery of 5-Fluorouracil Conjugated with Silkworm Pupa Derived Protein Nanoparticles for Experimental Lymphoma Cancer.

Protein-based drug delivery systems have an edge over conventional drug delivery systems due to their biodegradability, non-antigenicity, and excellent biocompatibility to improve the therapeutic properties of anticancer drugs. This study describes the increased anticancer efficacy of 5-fluorouracil (5-FU) conjugated with silkworm Bombyx mori pupal biowaste derived nanoparticles. Here, we have checked the toxicity of pupa-protein nanoparticles (PpNps) and their potential as a carrier for anticancer drugs. PpNps were prepared by a desolvation method which resulted in a uniform particle size of 162.7 ± 2.9 nm. The 5-FU loaded PpNps were formulated and characterized. The drug content of the developed 5-FU conjugated nanoparticles was evaluated by HPLC analysis. The entrapment efficiency and loading capacity of 5-FU were analyzed by HPLC and determined to be 93% and 88.6%, respectively. The release studies showed the biphasic release of 5-FU at pH 7.4 where rapid drug release was achieved for first 30 min, followed by a sustained release of 5-FU from the developed Nps achieved for the next 8 h. Mice with developed ascites tumors were intraperitoneally treated with 5-FU-PpNps and sacrificed. There was a significant increase in total red blood cells and hemoglobulin in 5-FU-PpNps treated mice, whereas a significant decrease in white blood cells which indicated the reduced inflammation of cancer. Subsequently, 5-FU-PpNps decreased the tumor volume and tumor cell viability, which proved its cytotoxic property to cancer cells. This study presents a novel approach to derive B. mori pupal protein nanoparticles, which can be safely used for cancer drug delivery.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection.

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.

A novel biological role for nsLTP2 from Oriza sativa: Potential incorporation with anticancer agents, nucleosides and their analogues

Development of a protein-based drug delivery system has major impact on the efficacy and bioavailability of unstable and water insoluble drugs. In the present study, the binding modes of a nonspecific lipid transfer protein (nsLTP2) from Oryza sativa with various nucleosides and analogous molecules were identified. The 3-D structure of the protein was designed and validated using modeler 9.13, Molegro virtual docker and procheck tool, respectively. The binding affinity and strength of interactions, key contributing residues and specificity toward the substrates were accomplished by computational docking and model prediction. The protein presented high affinity to acyclovir and vidarabine as purine-analogous drugs. Binding affinity is influenced by the core template and functional groups of the ligands which are structurally different cause the variation of interaction energies with nsLTP2. Nonetheless, all the evaluated analogous drugs occupy the proximity space at the nsLTP active site with high similarity in their binding modes. Our findings hold great promise for the future applications of nsLTPs in various aspects of pharmaceutical science and molecular biology.

Processing of gelatin-based cryogels with improved thermomechanical resistance, pore size gradient, and high potential for sustainable protein drug release.

Porous gelatin (GEL) cryogels were processed by spatiotemporal and temperature-controlled gelation and freezing-lyophilizaton process, followed by zero-length crosslinking, using different molarities of reagents (EDC and NHS) and reaction media (100% PBS or 20/80% PBS/EtOH mixture) for variable time extensions (1-24 h). In this way, tuneable cryogels with gradient microporosity (from 100 µm to 1000 µm) were formed, being mainly influenced by crosslinkers' concentration and EtOH addition. Later affect the pore morphology (from round to ellipsoid), consequently modulating the steady-state physiological swelling profile toward twice lower values (∼ 600%) comparing to stepwise swelling of in 100% PBS media crosslinked cryogels. While the presence of EtOH decelerate the crosslinking kinetic by retaining cryogels' microstructure formed during freezing, the 100% PBS and higher EDC molarity resulted in approximately 40% crosslinking degree, being expressed as a thermal resistance of cryogels up to approximately 73°C. Finally, the tuneable enzymatic resistance allow time-dependent poly-L-Lysine (pL) release profile in up to month period. The processed GEL cryogels have potential in broad range biomedical applications, especially as sustainable, protein-based drug delivery systems.

Photo synthesis of protein-based drug-delivery nanoparticles for active tumor targeting.

Recently, nanometer-sized drug-delivery systems (DDSs) with an active targeting function to tumor sites have been cinfirmed to have great efficiency for cancer chemotherapy. However, the use of complicated modification techniques and the poor graft efficiency of the targeting ligands on the surface of the DDS have largely limited the application of most of the nano-DDSs. Especially for protein-based nano-DDSs, grafting of targeting ligands often requires genetic modification of the protein sequences, which is laborious and inefficient. Here, we present a novel method to photo synthesize a protein-based drug-delivery system with an active targeting function to tumor sites. The disulfide bonds in protein bovine α-lactalbumin (BLA) can be ruptured by controlled UV illumination, which triggers the formation of nano-sized protein aggregates and releases free thiol groups for the modification of the active targeting ligand of circular RGD peptide. Moreover, we demonstrated that the anti-cancer drug doxorubicin (DOX) can be loaded onto the protein-based DDS during the photo synthesis step. The synthesis approach is very convenient and cost-effective and can be accomplished under physiological conditions. Both in vitro and in vivo experiments validate that this DDS system possesses a much greater drug-delivery efficiency to the tumor sites and a better inhibition capability of tumor growth than the unmodified counterparts. This novel drug-delivery system can find broad applications in cancer chemotherapy. Moreover, our synthetic strategy can be easily extended to other disulfide-containing proteins and greatly expand the tool-box of protein-based DDSs for active targeting.

Apoferritin-based nanomedicine platform for drug delivery: equilibrium binding study of daunomycin with DNA

This paper describes the preparation and optimization of the analytical properties of the protein based drug delivery platform apoferritin. In biological systems, the protein cage ferritin is used to store iron and to keep it from building to toxic levels in cells. When the iron atoms are removed from ferritin, apoferritin is formed. In this study, daunomycin, an anthracycline antibiotic drug that has been used for specific types of cancer treatment such as acute myeloid leukemia and acute lymphocytic leukemia, was encapsulated within the protein cage for drug delivery. Daunomycin slows or stops the growth of cancer cells by binding with the cell's DNA. The model for daunomycin-DNA complex binding mechanism is intercalation, where daunomycin binds with approximately every 3 base pairs causing a local unwinding, but a negligible distortion of the helical conformation. The binding affinity for free DNA is higher than that of structured DNA in cells. Upon binding with DNA the fluorescence intensity of daunomycin decreases. We used apoferritin's ability to disassemble and reassemble under pH control to load the therapeutic compound daunomycin. The combination of a modifiable interior and exterior surface and the passable hydrophobic and hydrophilic channels through the cage allows the containment or attachment of both insoluble and soluble drugs for delivery. At experimental pH 5 conditions the interaction between the apoferritin interior cage and daunomycin is weak making it difficult to encapsulate the drug effectively within the protein cage. The incorporation of poly-L-aspartic acid (PLAA), a polypeptide and biodegradable material that does not increase the toxicity of the drug delivery system and is negatively charged at pH 5.0, into the drug delivery system resulted in a substantial improvement in the drug encapsulation. The binding properties of free daunomycin with DNA were compared to the newly synthesized apoferritin protein based drug delivery system. Encapsulation of the daunomycin within the apoferritin protein cage had little effect upon the intrinsic binding constant, Ki, or the exclusion parameter n as compared to the free daunomycin model. The study resulted in the design and optimization of a unique protein based drug delivery platform using the protein cage apoferritin for potential therapeutic administration of the anti-cancer agent daunomycin.

Protein‐Based Nanomedicine Platforms for Drug Delivery

Protein-based nanomedicine platforms for drug delivery comprise naturally self-assembled protein subunits of the same protein or a combination of proteins making up a complete system. They are ideal for drug-delivery platforms due to their biocompatibility and biodegradability coupled with low toxicity. A variety of proteins have been used and characterized for drug-delivery systems, including the ferritin/apoferritin protein cage, plant-derived viral capsids, the small Heat shock protein (sHsp) cage, albumin, soy and whey protein, collagen, and gelatin. There are many different types and shapes that have been prepared to deliver drug molecules using protein-based platforms, including various protein cages, microspheres, nanoparticles, hydrogels, films, minirods, and minipellets. The protein cage is the most newly developed biomaterial for drug delivery and therapeutic applications. The uniform size, multifunctionality, and biodegradability push it to the frontier of drug delivery. In this Review, the recent strategic development of drug delivery is discussed with emphasis on polymer-based, especially protein-based, nanomedicine platforms for drug delivery. The advantages and disadvantages are also discussed for each type of protein-based drug-delivery system.