Photo-synthesis of protein-based nanoparticles and the application in drug delivery

Recently, protein-based nanoparticles as drug delivery systems have attracted great interests due to the excellent behavior of high biocompatibility and biodegradability, and low toxicity. However, the synthesis techniques are generally costly, chemical reagents introduced, and especially present difficulties in producing homogeneous monodispersed nanoparticles. Here, we introduce a novel physical method to synthesize protein nanoparticles which can be accomplished under physiological condition only through ultraviolet (UV) illumination. By accurately adjusting the intensity and illumination time of UV light, disulfide bonds in proteins can be selectively reduced and the subsequent self-assembly process can be well controlled. Importantly, the co-assembly can also be dominated when the proteins mixed with either anti-cancer drugs, siRNA, or active targeting molecules. Both in vitro and in vivo experiments indicate that our synthesized protein–drug nanoparticles (drug-loading content and encapsulation efficiency being ca. 8.2% and 70%, respectively) not only possess the capability of traditional drug delivery systems (DDS), but also have a greater drug delivery efficiency to the tumor sites and a better inhibition of tumor growth (only 35% of volume comparing to the natural growing state), indicating it being a novel drug delivery system in tumor therapy.     

Controlled self assembly of collagen nanoparticle

In recent years carrier-mediated drug delivery has emerged as a powerful methodology for the treatment of various pathologies. The therapeutic index of traditional and novel drugs is enhanced via the increase of specificity due to targeting of drugs to a particular tissue, cell or intracellular compartment, the control over release kinetics, the protection of the active agent, or a combination of the above. Collagen is an important biomaterial in medical applications and ideal as protein-based drug delivery platform due to its special characteristics, such as biocompatibility, low toxicity, biodegradability, and weak antigenicity. While some many attempts have been made, further work is needed to produce fully biocompatible collagen hydrogels of desired size and able to release drugs on a specific target. In this article we propose a novel method to obtain spherical particles made of polymerized collagen surrounded by DMPC liposomes. The liposomes allow to control both the particles dimension and the gelling environment during the collagen polymerization. Furthermore, an optical based method to visualize and quantify each step of the proposed protocol is detailed and discussed.     

具有双重治疗机制的磁性纳米无定型氧化铁基药物递送系统

合成一种具有pH响应性的聚乙二醇(PEG)修饰无定形介孔氧化铁纳米粒子(AFe-PEG). 这种纳米粒子可以高效负载药物分子如阿霉素(DOX),构成新型多功能AFe-PEG/DOX药物递送体系. DOX的负载率高达948 mg/g-纳米粒子. 在酸性溶液中,AFe-PEG/DOX纳米粒子不仅可以有效释放DOX,同时可以释放Fe离子进行Fenton反应,将H₂O₂转变成·OH自由基. 体外实验结果表明,AFe-PEG/DOX纳米粒子对HeLa细胞同时具有化疗和化学动力学疗法的疗效. 同时,由于AFe-PEG/DOX 纳米粒子本身的磁性,使其在外部磁场中的细胞内化效率也得到了提高.     

Utilizing inverse micelles to synthesize calcium phosphate nanoparticles as nano-carriers

The aim of this study was to investigate the feasibility of the inverse micelles (IM) technique in producing protein-loaded calcium phosphate nanoparticles (CaP NPs), and to compare this technique with the conventional co-precipitation (co-ppt) technique. In this study, bovine serum albumin and lysozyme were used as model proteins. The results show that CaP NPs produced by IM were shown to be spherical and homogenous in size of ~50 nm. Protein loading efficiency of the IM technique was shown to be much higher than CaP NPs synthesized through co-ppt technique. X-ray photoelectron spectroscopy shows that proteins were not adsorbed onto the surface of IM-synthesized CaP NPs, which suggested that the proteins were entrapped within the particle matrix. Release studies show that protein release was more rapid at lower pH conditions (pH 5 and 6) than at physiological pH of 7.4. A burst release was observed for co-ppt CaP NPs, while a continuous release of protein was observed for IM-produced CaP NPs. This study shows the superiority of the IM technique in preparing pH responsive CaP NPs as nano-carriers.     

Selective Enrichment of Polydopamine in Mesoporous Nanocarriers for Nuclear‐Targeted Drug Delivery

Small particle size and strong host–guest interactions are prerequisites in the field of nuclear‐targeting nanocarriers for overcoming the multidrug resistance of cancer cells. A novel scheme of synthesizing hybrid organic–inorganic nanocarriers with mesopores is introduced to enhance the delivery efficiency of therapeutic drugs. Specifically, inorganic silica and organic polydopamine (PDA) are integrated inside the pore framework by the assistance of organic silanes terminated by amino/thiol groups. Silica‐etching by hydrothermal treatment leads to the selective enrichment of bioadhesive PDA and size reductions for the hybrids (to ≈30 nm). Interestingly, a high drug loading capacity (523 µg mg⁻¹ for doxorubicin hydrochloride), as well as pH/ glutathione dual‐responsive drug release properties, are realized by the nanocarriers, owing to their high surface area (825 m² g⁻¹) and the π‐stacking and/or hydrophobic–hydrophobic interactions stemming from PDA. More importantly, the conjugation of TAT peptide facilitates the intranuclear localization of the nanocarriers and the release of the encapsulated drugs directly within the nucleoplasm of the multidrug resistant MCF‐7/ADR cancer cells. Therefore, these results provide a controllable method of engineering high‐surface‐area nanocarriers with bioadhesive polymers on the pore surface for advanced drug delivery applications.     

A Functionalized Hollow Mesoporous Silica Nanoparticles‐Based Controlled Dual‐Drug Delivery System for Improved Tumor Cell Cytotoxicity

Multifunctional nanoparticles for selectively targeting tumor cells and effectively delivering multiple drugs are urgently needed in cancer therapy. Here, a dual‐drug delivery system is prepared, based on functionalized hollow mesoporous silica nanoparticles (HMSNs). Doxorubicin (DOX) hydrochloride is loaded into the hollow core, and dichloro(1,2‐diaminocyclohexane)platinum (II) (DACHPt) is stored in the pores of the shell by the coordination interaction with the carboxyl groups modified on the pore walls, which also serves as barriers to control the DOX release. Detailed studies in vitro indicate that the DACHPt release is triggered by Cl^? through the cleavage of the coordination interaction, and the DOX release depends on the release rate of DACHPt and the environmental pH value. The surface of the mechanized nanoparticles is also modified by transferrin (Tf) to achieve the tumor specificity. Compared with individual drug delivery systems, the dual‐drug delivery system shows synergistic efficacy on the cell cytotoxicity (combination index = 0.30), resulting in improved tumor cell killing. The present dual‐drug delivery system provides a promising strategy to develop controlled and targeted combination therapies for efficient cancer treatment.     

Polysaccharides-based polyelectrolyte nanoparticles as protein drugs delivery system

Polysaccharides-based nanoparticles were prepared by synthesized quaternized chitosan and dextran sulfate through simple ionic-gelation self-assembled method. Introduction of quaternized groups was intended to increase water solubility of chitosan and make the nanoparticles have broader pH sensitive range which can remain more stable in physiological pH and decrease the loss of protein drugs caused by the gastric cavity. The load of BSA was affected by molecular parameter, i.e., degree of substitution, and average molecular weight of quaternized chitosan, as well as concentration of BSA. Fast release occurred in phosphate buffer solution (pH 7.4) while the release was slow in hydrochloric acid (pH 1.4). The drug release mechanism is Fickian diffusion through release kinetics analysis. Cell uptake demonstrated nanoparicles can internalize into Caco-2 cells, which suggested that nanoparticles had good biocompatibility. No significant conformation change was noted for the released BSA in comparison with native BSA using circular dichroism spectroscopy. This kind of novel composite nanoparticles may be a promising delivery system for oral protein and peptide drugs.     

Controlled oxidation of iron nanoparticles in chemical vapour synthesis

Enteric-coated formulations can delay the release of drugs until they have passed through the stomach. However, high concentration of drugs caused by rapidly released in the small intestine leads to the intestinal damage, and frequent administration would increase the probability of missing medication and reduce the patient compliance. To solve the above-mentioned problems, aspirin-loaded enteric-coated sustained-release nanoparticles with core–shell structure were prepared via one-step method using coaxial electrospray in this study. Eudragit L100-55 as pH-sensitive polymer and Eudragit RS as sustained-release polymer were used for the outer coating and inner core of the nanoparticles, respectively. The maximum loading capacity of nanoparticles was 23.66 % by changing the flow rate ratio of outer/inner solutions, and the entrapment efficiency was nearly 100 %. Nanoparticles with core–shell structure were observed via fluorescence microscope and transmission electron microscope. And pH-sensitive and sustained drug release profiles were observed in the media with different pH values (1.2 and 6.8). In addition, mild cytotoxicity in vitro was detected, and the nanoparticles could be taken up by Caco-2 cells within 1.0 h in cellular uptake study. These results indicate that prepared enteric-coated sustained-release nanoparticles would be a more safety and effective carrier for oral drug delivery.     

Positively charged biopolymeric nanoparticles for the inhibition of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> biofilms

Currently, many microbial infections have the potential to become lethal owing to the development of antimicrobial resistance by means of different mechanisms and mainly on the basis of the fact that many drugs are unable to reach therapeutic levels in the target sites. This requires the use of high doses and frequent administrations, causing adverse side effects or in some cases toxicity. The use of nanoparticle systems could help overcome such problems and increase drug efficacy. In the present study, we developed a new drug delivery system based on the use of biopolymeric nanovectors loaded with tobramycin (Tb), which is the standard antibiotic for the treatment of Cystic Fibrosis-associated P. aeruginosa lung infections. Tb-loaded biopolymeric nanoparticles composed by dextran sulfate (DS) and chitosan (CS) were prepared by ionotropic gelation. We optimized drug entrapment in DS/CS nanoparticles, obtaining particles of 170 nm and with a drug loading of 400 µg Tb/mg of nanoparticles. In accord with in vitro release experiments, such preparations were able to release approximately 25 % of their cargo in 60 h. In vitro, the antimicrobial efficacy of the drug delivery system on P. aeruginosa biofilm was tested and compared to the effects of free drug revealing that this formulation can reduce the viability of P. aeruginosa biofilms for 48 h with a single-dose administration.     

Cavitation technology – A greener processing technique for the generation of pharmaceutical nanoemulsions

Novel nanoemulsion-based drug delivery systems (DDS) have been proposed as alternative and effective approach for the delivery of various types of poorly water-soluble drugs in the last decade. This nanoformulation strategy significantly improves the cell uptake and bioavailability of numerous hydrophobic drugs by increasing their solubility and dissolution rate, maintaining drug concentration within the therapeutic range by controlling the drug release rate, and reducing systemic side effects by targeting to specific disease site, thus offering a better patient compliance. To date, cavitation technology has emerged to be an energy-efficient and promising technique to generate such nanoscale emulsions encapsulating a variety of highly potent pharmaceutical agents that are water-insoluble. The micro-turbulent implosions of cavitation bubbles tear-off primary giant oily emulsion droplets to nano-scale, spontaneously leading to the formation of highly uniform drug contained nanodroplets. A substantial body of recent literatures in the field of nanoemulsions suggests that cavitation is a facile, cost-reducing yet safer generation tool, remarkably highlighting its industrial commercial viability in the development of designing novel nanocarriers or enhancing the properties of existing pharmaceutical products. In this review, the fundamentals of nanoemulsion and the principles involved in their formation are presented. The underlying mechanisms in the generation of pharmaceutical nanoemulsion under acoustic field as well as the advantages of using cavitation compared to the conventional techniques are also highlighted. This review focuses on recent nanoemulsion-based DDS development and how cavitation through ultrasound and hydrodynamic means is useful to generate the pharmaceutical grade nanoemulsions including the complex double or submicron multiple emulsions.     

Design of a Novel Mesoporous Organosilica Hybrid Microcarrier: a pH Stimuli‐Responsive Dual‐Drug‐Delivery Vehicle for Intracellular Delivery of Anticancer Agents

The integration of unique functionality into mesoporous organosilica hybrid carriers is an important issue in solving the challenges of dual/multi delivery for combined therapy with drugs with a distinct therapeutic effects. Newly designed mesoporous organosilica hybrid microcarriers (HMCs) are synthesized on the basis of the triblock‐copolymer‐templated sol–gel method. The synthesized HMCs, which integrate both heteroaromatic pyridine and diurea functionalities, are combined in a mesoporous organosilica hybrid network to design functional hybrid microcarriers with a range of mechanisms for the pH‐triggered release of two drugs. The drugs include the hydrophilic anticancer therapeutic agent 5‐fluorouracil (5‐FU) and the non‐steroidal hydrophobic anti‐inflammatory drug ibuprofen (IBU). 5‐FU and IBU are encapsulated in the HMCs using multiple hydrogen bonding and electrostatic interaction sites and are delivered under a range of pH conditions. The release of 5‐FU and IBU is tested at pH 5.5 and 7.4. The results show that the release is sensitive to pH. The antitumor activity of the released 5‐FU is evaluated using the MCF‐7 cell line. The released 5‐FU has the capacity to kill cancer cells under acidic pH conditions.     

Colloidosomes as Single Implantable Beads for the In Vivo Delivery of Hydrophobic Drugs

The synthesis of silica‐based colloidosomes with a polymer core obtained via inverse Pickering emulsification and their use as an implantable drug delivery system in zebrafish are described. Silica nanoparticles act as a stabilizer of a water‐in‐oil emulsion creating aqueous droplets with a silica shell. The core of the colloidosomes is polymerized resulting in tough particles. Colloidosomes loaded with model drugs show a release profile dependent on the porosity of the silica nanoparticles. Studying the effect of drugs on zebrafish development and tail regeneration is a new and emerging field in biomedical research. The in vivo delivery and bioactivity of retinoic acid from single implanted colloidosomes in partially amputated caudal fins are shown at the phenotype and genotype level. The colloidosomes are biocompatible since no signs of inflammation are observed. With these initial studies, the use of colloidosomes as single implantable beads is demonstrated for the local in vivo release of bioactive drugs. It is envisioned that these single particles can be applied for a broad range of hydrophobic drugs.     

Magnetic nanoemulsions as drug delivery system for Foscan: Skin permeation and retention in vitro assays for topical application in photodynamic therapy (PDT) of skin cancer

In this work, we performed the synthesis and in vitro characterization of a new class of drug delivery system (DDS) denominated magnetic nanoemulsion (MNE). The association of colloidal nanoparticles with biocompatible magnetic fluids results in a new DDS for application in photodynamic therapy (PDT) and magnetic hyperthermia treatment. It works in a synergic manner with an expected enhancement in tumor damage after minimum drug doses, based on heat dissipation and/or light photosensitization. For this purpose, we investigated the permeation and retention in vitro model using Foscan^® as a photosensitizer incorporated in MNE using a Franz diffusion cell and a biological skin model in biomimetic conditions.     

Drug release from various thicknesses of layered mats consisting of electrospun polycaprolactone and polyethylene oxide micro/nanofibers

A new drug delivery system (DDS) consisting of electrospun nanofibers is proposed. Layered mats of hydrophobic polycaprolactone (PCL) and polyethylene oxide (PEO) nanofibers were prepared successfully in a layer-by-layer manner using an electrospinning process. The PEO mat and drug were co-electrospun as a drug reservoir. Drug release rate was controlled physically by the thickness of the electrospun nanofibrous PCL layer, and its release behavior was examined over time. Release tests showed that the release behavior and the amount of initial burst of the drug were critically dependent on the thickness of the nanofibrous PCL mat. The release of drug showed a linear relationship with the thickness of the porous electrospun PCL mat. In addition, to demonstrate the feasibility of this type of DDS, the release behavior of the antimicrobial peptide HPA3NT3 from the nanofiber system was examined. The release of the peptide was easily controlled by the PCL nanofiber thickness and the released peptide did not lose biological activity.     

A General Strategy to Encapsulate Semiconducting Polymers within PEGylated Mesoporous Silica Nanoparticles for Optical Imaging and Drug Delivery

Although semiconducting polymers (SPs) have become an important category for optical imaging and phototherapy, their biomedical application is still facing a number of challenges. Herein, a cationic surfactant–assisted approach to encapsulate hydrophobic SPs within highly PEGylated mesoporous silica (mSiO₂) nanoparticles with excellent colloidal stability and enhanced fluorescence in aqueous solution is reported. In comparison to the previously reported amphiphilic polymer coating and silification method, this universal strategy not only suppresses the formation of empty polymer micelles and free silica nanoparticles, but also provides high specific surface area for drug loading. As a proof of concept, furan-containing diketopyrrolopyrrole-based semiconducting polymers (PDFT) are coated with mesoporous silica and utilized for fluorescence imaging in the second near-infrared region (NIR-II, 1000–1700 nm) and drug delivery. In vivo blood vessel imaging and tumor imaging are achieved with high resolution (0.21 mm) and signal-to-background ratio (≈4.2). Additionally, pH-responsive drug release and improved therapeutic effect are observed. By choosing desired SPs, different optical imaging and therapeutic modalities can also be achieved, thus the SP@mSiO₂ nanostructures obtained here provide numerous opportunities for theranostic applications.     

Dual Responsive Poly(N‐vinylcaprolactam) Based Degradable Microgels for Drug Delivery

Poly(N‐vinylcaprolactam)‐based biodegradable microgels are prepared for drug delivery application via precipitation polymerization using diacetone acrylamide (DAAM) and dimethyl itaconate (IADME) as comonomers. The microgel particles are subsequently crosslinked by addition of adipic acid dihydrazide, which reacts with the ketone groups of DAAM. Itaconic acid (IA) groups are generated by the hydrolysis of IADME units inside the microgels resulting into both pH and temperature sensitive microgel particles. Volume phase transition temperature of the obtained microgels is influenced by both IA content and pH of the surrounding medium. Due to the incorporation of hydrazone linkages, the microgels show degradation under acidic conditions. These microgels can effectively encapsulate doxorubicin (DOX) as a model drug and show low DOX leakage under physiological conditions while rapid DOX release is observed at low pH. The results of the cytotoxicity assay further display that the DOX‐loaded microgels exhibit effective antitumor activity against HeLa cells demonstrating their great potential as drug delivery carriers for cancer therapy.     

GLUT1-Targeting and GSH-Responsive DOX/L61 Nanodrug Particles for Enhancing MDR Breast Cancer Therapy

Efficient targeting to tumor tissues and subsequent rapid drug release in cancer cells remains a major challenge for nanodrug delivery systems. Herein, smart nanodrug particles with reduction-sensitive and active tumor-targeting ability are constructed based on the nanoprecipitation of glucosamine-grafted pluronic L61 (GA-L61) and disulphide-linked doxorubicin dimer (DOX SS DOX) to overcome tumor multidrug resistance (MDR). These nanoparticles show proper size and excellent stability under neutral conditions, while quickly release DOX due to the breakage of disulfide bonds under reductive medium. In vitro cellular uptake and drug efflux demonstrate that L61 can efficiently increase DOX concentration in MCF/ADR resistant cells by inhibiting the function of drug resistance proteins. In vivo biodistribution reveals that glucose transporter 1 (GLUT1)-mediated tumor-targeting significantly improves tumor accumulation of the glucosamine-contained nanoparticles. Finally, the combination of GLUT1-targeting, glutathione (GSH)-responsive, and MDR-reversal effects in nanoparticles achieve superior antitumor effects, which can provide an efficient, safe, and economic approach for drug delivery and cancer chemotherapy.     

Self‐Assembly of Drug–Drug Conjugates as Drug Self‐Delivery System for Tumor‐Specific pH‐Triggered Release

An acid‐labile doxorubicin dimer (D‐DOX) is designed as drug–drug conjugate for tumor intracellular pH‐triggered release, by conjugating doxorubicin (DOX) with adipic acid dihydrazide (ADH). The dimer‐based surfactants modified with polyethylene glycol (PEG), DOX‐ADH‐DOX‐PEG or are synthesized by mono‐PEGylation and bi‐PEGylation, respectively. Then the prodrug nanoparticles are fabricated with different drug contents via dialyzing the mixture solution of D‐DOX and the PEGylated surfactants in dimethyl sulfoxide (DMSO) with different mass ratios against water. It is found that the smaller prodrug nanoparticles (142–163 nm) could be obtained with the mono‐PEGylated surfactant, than those of 157–225 nm with the bi‐PEGylated surfactant. Furthermore, the mono‐PEGylated surfactant results in a higher drug content of 51% due to their lower PEG contents. All prodrug nanoparticles could release DOX completely within 36 h at pH 5.0, with the premature drug leakage of less than 10% at pH 7.4. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays demonstrate the proposed drug self‐delivery system possessed an enhanced anticancer efficacy against HepG2 cells than the free DOX.     

Nanoscale Janus Particles with Dual Protein Functionalization

Biofunctionalized Janus particles with tailored surface chemistry are gathering interest for applications as catalysts, multifunctional cell surface targets, nanomotors, and drug delivery systems. The dual nature of the surface chemistry of Janus particles can be exploited to immobilize drugs, cell surface targets, and/or other functional molecules on both sides of the particle surface. In this study, a model system is established for the scalable preparation of nanoscale Janus particles with dual protein functionalization with the proteins ferritin and streptavidin. 80 nm silica nanoparticles (SiNPs) modified with azidosilane are used to prepare Pickering emulsions with molten wax as the droplet phase. The azide‐functionalized SiNPs on the Pickering emulsion droplets are further subjected to face‐selective silanization with biotin‐polyethylene glycol ethoxy silane. Afterward, ferritin is grafted on the azide‐functionalized side via a click‐reaction and the biotin groups are conjugated with streptavidin which is labeled with ultrasmall gold nanoparticles. In order to elucidate the advantages and limits of this approach, a detailed characterization is performed of the particles at every process step. The results show that this method represents a scalable platform for the versatile preparation of nanoscale Janus nanoparticles that can potentially be used with a wide variety of proteins.     

Polymer Capsules as a Theranostic Tool for a Universal In Vitro Screening Assay—The Case of Lysosomal Storage Diseases

Lysosomal storage disorders are rare genetic diseases characterized by a lysosomal enzyme deficiency. The defect leads to an accumulation of normally degraded substrates within the lysosomes. The accumulation of polymeric capsules inside the lysosomes is exploited to create a universal in vitro theranostic tool for lysosomal storage disorders. The diagnostic ability of this tool based on pH‐sensitive fluorophores is demonstrated by monitoring the lysosomal pH in Krabbe‐disease cell models upon accumulation of the substrate psychosine. Krabbe‐affected cells maintain their normal pH, while the lysosomes of their healthy counterparts undergo alkalinization, which can be correlated to toxicity. The potential of this tool for therapy based on enzymes inside the capsules is evaluated within the context of enzyme replacement therapy. Enzymatic degradation of the capsules inside the lysosome leads to release of the encapsulated active enzyme and the prevention of adverse effects of accumulated psychosine upon capsule‐based delivery of the functional enzyme is confirmed. In Fabry‐affected cells the intracellular enzymatic activity of the drug Replagal released from capsules shows the same kinetics as the free enzyme, which constitutes the current therapy, although the activity is smaller. Encapsulating Replagal nevertheless represents an alternative to receptor‐mediated endocytosis, overcoming limitations such as low or absent receptor expression.