Surface engineering of gold nanoparticles for in vitro siRNA delivery

Cellular uptake, endosomal/lysosomal escape, and the effective dissociation from the carrier are a series of hurdles for specific genes to be delivered both in vitro and in vivo. To construct siRNA delivery systems, poly(allylamine hydrochloride) (PAH) and siRNA were alternately assembled on the surface of 11.8 ± 0.9 nm Au nanoparticles (GNP), stabilized by denatured bovine serum albumin, by the ionic layer-by-layer (LbL) self-assembly method. By manipulating the outmost PAH layer, GNP-PAH vectors with different surface electric potentials were prepared. Then, the surface potential-dependent cytotoxicity of the resultant GNP-PAH particles was evaluated via sulforhodamine B (SRB) assay, while the surface potential-dependent cellular uptake efficiency was quantitatively analyzed by using the flow cytometry method based on carboxyfluorescein (FAM)-labeled siRNA. It was revealed that the GNP-PAH particles with surface potential of +25 mV exhibited the optimal cellular uptake efficiency and cytotoxicity for human breast cancer MCF-7 cells. Following these results, two more positively charged polyelectrolytes with different protonating abilities in comparison with PAH, i.e., polyethylenimine (PEI), and poly(diallyl dimethyl ammonium chloride) (PDDA), were chosen to fabricate similarly structured vectors. Confocal fluorescence microscopy studies indicated that siRNA delivered by GNP-PAH and GNP-PEI systems was better released than that delivered by the GNP-PDDA system. Further flow cytometric assays based on immunofluorescence staining of the epidermal growth factor receptor (EGFR) revealed that EGFR siRNA delivered by GNP-PAH and GNP-PEI exhibited similar down-regulation effects on EGFR expression in MCF-7 cells. The following dual fluorescence flow cytometry assays by co-staining phosphatidylserine and DNA suggested the EGFR siRNA delivered by GNP-PAH exhibited an improved silencing effect in comparison with that delivered by the commercial transfection reagent Lipofectamine 2000.     

Multifunctional nanogels for siRNA delivery

The application of RNA interference to treat disease is an important yet challenging concept in modern medicine. In particular, small interfering RNA (siRNA) have shown tremendous promise in the treatment of cancer. However, siRNA show poor pharmacological properties, which presents a major hurdle for effective disease treatment especially through intravenous delivery routes. In response to these shortcomings, a variety of nanoparticle carriers have emerged, which are designed to encapsulate, protect, and transport siRNA into diseased cells. To be effective as carrier vehicles, nanoparticles must overcome a series of biological hurdles throughout the course of delivery. As a result, one promising approach to siRNA carriers is dynamic, versatile nanoparticles that can perform several in vivo functions. Over the last several years, our research group has investigated hydrogel nanoparticles (nanogels) as candidate delivery vehicles for therapeutics, including siRNA. Throughout the course of our research, we have developed higher order architectures composed entirely of hydrogel components, where several different hydrogel chemistries may be isolated in unique compartments of a single construct. In this Account, we summarize a subset of our experiences in the design and application of nanogels in the context of drug delivery, summarizing the relevant characteristics for these materials as delivery vehicles for siRNA. Through the layering of multiple, orthogonal chemistries in a nanogel structure, we can impart multiple functions to the materials. We consider nanogels as a platform technology, where each functional element of the particle may be independently tuned to optimize the particle for the desired application. For instance, we can modify the shell compartment of a vehicle for cell-specific targeting or evasion of the innate immune system, whereas other compartments may incorporate fluorescent probes or regulate the encapsulation and release of macromolecular therapeutics. Proof-of-principle experiments have demonstrated the utility of multifunctional nanogels. For example, using a simple core/shell nanogel architecture, we have recently reported the delivery of siRNA to chemosensitize drug resistant ovarian cancer cells. Ongoing efforts have resulted in several advanced hydrogel structures, including biodegradable nanogels and multicompartment spheres. In parallel, our research group has studied other properties of the nanogels, including their behavior in confined environments and their ability to translocate through small pores.     

Recent advances in the design,development, and targeting mechanisms of polymeric micelles for delivery of siRNA in cancer therapy

Small interfering RNA (siRNA) is a relatively novel nucleic acid-based therapy to treat diseases such as cancer. Nevertheless, substantial obstacles to its clinical applications have been reported, such as low cellular uptake, immunogenicity, off-target effects, and instability in physiological environments. The design of appropriate delivery vehicles capable of transporting siRNA to target cells has been pursued. Nanoparticles are extensively studied for the delivery of siRNA. Among the various nanocarriers, polymeric micelles have recently gained strong interest. Polymeric micelles of average nanometer size are straightforward to design and modify. Hydrophilic groups incorporated in the polymeric micelles can extend in vivo half-life of siRNA to ensure adequate accumulation in tumors, be exchanged for cations that electrostatically interact with siRNA, and be coupled to various ligands for cell-specific targeting. The polymeric micelle core provides stability and serves as a loading dock for drugs. In this review, the different types of polymers used, the design and characterization of polymeric micelles for siRNA delivery, and the established polymeric micelle targeting mechanisms are discussed.     

Hydrogels in drug delivery: Progress and challenges

There has been considerable progress in recent years in addressing the clinical and pharmacological limitations of hydrogels for drug delivery applications but substantial challenges remain. Here we discuss recent progress in overcoming these challenges, particularly with regards to effectively delivering hydrogels inside the body without implantation, prolonging the release kinetics of drugs from hydrogels, and expanding the nature of drugs which can be delivered using hydrogel-based approaches.     

Depolymerized chitosan nanoparticles for protein delivery: Preparation and characterization

In this article we describe our preliminary work involving the use of depolymerized, low molecular weight chitosan nanoparticles as carriers for proteins and peptides. We hypothesized that the molecular weight of chitosan could favorably modulate the particle and protein release characteristics for the delivery of certain bioactive macromolecules. Our primary objectives were to develop nanoparticle formulations that were stable and reproducible across a range of chitosan molecular weights and then characterize the physicochemical and in vitro release properties as functions of the polymer size. Using depolymerized fragments generated by NaNO₂ degradation of different chitosan salts, we prepared nanoparticle formulations based on ionotropic gelation with sodium tripolyphosphate (TPP). Regardless of the formulation, the nanoparticle size decreased with decreasing molecular weight and the ζ‐potential values remained unchanged. Similar comparisons were made with the encapsulation of insulin and tetanus toxoid as model proteins. The results indicated that the quantity of TPP in a given formulation has a greater effect on the protein encapsulation than the chitosan molecular weight. In fast release environments (i.e., buffered media), there was no significant molecular weight effect that could be discerned. These data lead to the conclusion that, under these experimental conditions, the chitosan molecular weight has a measurable effect on the particle properties, although this effect is modest relative to other formulation parameters (e.g., TPP content, type of protein loaded). Because these subtle differences could have dramatic effects physiologically, work is currently underway to elucidate the possible applications of depolymerized chitosans for peptide delivery in vivo. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 12: 2769–2776, 2003     

Water-soluble hypocrellin A nanoparticles as a photodynamic therapy delivery system

A novel delivery system for the hydrophobic photosensitizer, hypocrellin A, has been developed that uses hypocrellin A nanoparticles prepared by a reprecipitation method. In comparison to the unmodified photosensitizer, the hypocrellin A nanoparticles not only possess superior water solubility, higher water and light stability but also display high singlet oxygen production and DNA photocleavage capability.     

抑制癌症治疗多药耐药性的纳米药物递送体系

多药耐药(Multi-drug resistance，MDR)是指肿瘤细胞对传统和新型化疗药物产生耐药性的现象，成为治疗恶性肿瘤的重要障碍。近年来，人们发现纳米药物递送体系(Nanoscale drug delivery systems ，NDDS)在抑制肿瘤多药耐药性方面表现出其独特优势，包括增加药物的溶解度和稳定性，避开药物外排泵、以更低的剂量和更少的副作用实现目标部位更高的药物浓度。因此，NDDS成为克服癌症MDR的有效策略。在这里，我们总结了近几年提高抗癌药物摄取和选择性细胞内蓄积的NDDS的种类及研究进展，包括靶向特异性配体功能化体系和刺激响应型药物的激活释放体系等。     

Surface engineering of iron oxide nanoparticles for targeted cancer therapy

Nanotechnology provides a flexible platform for the development of effective therapeutic nanomaterials that can interact specifically with a target in a biological system and provoke a desired response. Of the nanomaterials studied, iron oxide nanoparticles have emerged as one of top candidates for cancer therapy. Their intrinsic superparamagnetism enables noninvasive magnetic resonance imaging (MRI), and their biodegradability is advantageous for in vivo applications. A therapeutic superparamagnetic iron oxide nanoparticle (SPION) typically consists of three primary components: an iron oxide nanoparticle core that serves as both a carrier for therapeutics and contrast agent for MRI, a coating on the iron oxide nanoparticle that promotes favorable interactions between the SPION and the biological system, and a therapeutic payload that performs the designated function in vivo. Often, the design may include a targeting ligand that recognizes the receptors over-expressed on the exterior surface of cancer cells. The body is a highly complex system that imposes multiple physiological and cellular barriers to foreign objects. Thus, the success of a therapeutic SPION largely relies on the design of the iron oxide core to ensure its detection in MRI and the coatings that allow the nanoparticles to bypass these barriers. Strategies to bypass the physiological barriers, such as liver, kidneys, and spleen, involve tuning the overall size and surface chemistry of the SPION to maximize blood half-life and facilitate the navigation in the body. Strategies to bypass cellular barriers include the use of targeting agents to maximize uptake of the SPION by cancer cells and the employment of materials that promote desired intracellular trafficking and enable controlled drug release. The payload can be genes, proteins, chemotherapy drugs, or a combination of these molecules. Each type of therapeutic molecule requires a specific coating design to maximize the loading and to achieve effective delivery and release. In this Account, we discuss the primary design parameters in developing therapeutic SPIONs with a focus on surface coating design to overcome the barriers imposed by the body's defense system. We provide examples of how these design parameters have been implemented to produce SPIONs for specific therapeutic applications. Although there are still challenges to be addressed, SPIONs show great promise in the successful diagnosis and treatment of the most devastating cancers. Once the critical design parameters have been optimized, these nanoparticles, combined with imaging modalities, can serve as truly multifunctional theranostic agents that not only perform a therapeutic function but also provide instant clinical feedback, allowing the physician to adjust the treatment plan.     

Surface‐functionalized polyurethane nanoparticles for targeted cancer therapy

Polymer nanoparticles (nps) have gained growing interest as carriers for anticancer drugs as they can target tumour tissues by both passive and active pathways. While the passive targeting mechanisms mainly rely on the small size of the carriers, active targeting requires surface modifications of the polymer core in order to introduce specific functionalities to actively recognize cancer cells. The present work proposes an innovative method for the preparation of surface‐functionalized nps based on the use of biodegradable polyester‐ and polyester/ether‐urethanes (PURs) embedding amino functionalities. Two polyurethanes were prepared, one based on just poly(?‐caprolactone) diol (PCL‐PUR) and the other based on both PCL diol and poly(ethylene glycol) (PEG) (70/30 ratio, PCL‐PEG‐PUR). Nanoparticles of small size ranging between 150 and 200 nm and negative ζ potential (ranging from ?18 mV to ?27 mV) were obtained. Functional groups were exposed post nps preparation as confirmed by X‐ray photoelectron spectroscopy, ninhydrin assay and ¹H NMR, which evidenced a 24% tert‐butyloxycarbonyl cleavage for PCL‐PUR‐NH₂ nps and 29% for PCL‐PEG‐PUR‐NH₂ nps. The monoclonal antibody Herceptin (HER), which targets HER‐2 receptors, was coupled through ethyl(dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide (EDC/NHS) mediated chemistry. The optimal HER:NH₂ ratio was determined to be 1:16 for the PEG‐containing PUR and 1:8 for PCL‐PUR. HER‐nps maintained the intrinsic cytotoxicity of the antibody, as shown by the ca 50% decrease of HER‐2‐expressing HeLa cell viability. The results indicate that our protocol for surface functionalization of PUR nps, based on surface exposure of previously inserted functional groups followed by covalent coupling of biomolecules, is suitable for the preparation of nps for active recognition of target cells. © 2016 Society of Chemical Industry     

Diatomite silica nanoparticles for drug delivery

Diatomite is a natural fossil material of sedimentary origin, constituted by fragments of diatom siliceous skeletons. In this preliminary work, the properties of diatomite nanoparticles as potential system for the delivery of drugs in cancer cells were exploited. A purification procedure, based on thermal treatments in strong acid solutions, was used to remove inorganic and organic impurities from diatomite and to make them a safe material for medical applications. The micrometric diatomite powder was reduced in nanoparticles by mechanical crushing, sonication, and filtering. Morphological analysis performed by dynamic light scattering and transmission electron microscopy reveals a particles size included between 100 and 300 nm. Diatomite nanoparticles were functionalized by 3-aminopropyltriethoxysilane and labeled by tetramethylrhodamine isothiocyanate. Different concentrations of chemically modified nanoparticles were incubated with cancer cells and confocal microscopy was performed. Imaging analysis showed an efficient cellular uptake and homogeneous distribution of nanoparticles in cytoplasm and nucleus, thus suggesting their potentiality as nanocarriers for drug delivery.

PACS

87.85.J81.05.Rm; 61.46. + w     

Polycaprolactone-based biomaterials for tissue engineering and drug delivery: Current scenario and challenges

Recently, poly (?-caprolactone) (PCL) has gained a lot of attention, and shown great potential in biomedical applications. Among synthetic polymers, PCL is one of the easiest to process and manipulate into a large range of shapes and sizes due to its low melting temperature and its superior viscoelastic properties. In this review article the authors focus mainly on the properties of PCL-based biomaterials relevant to drug delivery and tissue engineering applications. The authors provide an insight into the recent developments and challenges of PCL-based biomaterials as a critical component of new therapeutic strategies for many diseases.     

Responsive polymeric nanoparticles for controlled drug delivery

Small‐molecule drugs often have limited solubility, display rapid clearance or poor selectivity that leads to undesired side‐effects. Although prodrug strategies can improve solubility and lower toxicity, activation ‘on demand’ as well as targeted transport of prodrugs remains a challenge in drug delivery. Responsive polymeric nanoparticles can help meet these challenges with the encapsulation or conjugation of drugs, allowing release at the target site upon triggering by an internal or external stimulus. The adaptable design of polymeric nanoparticles allows them to play a vital role in achieving a specific and desired response following application of a specific stimulus. Here, the most recent progress in responsive polymeric nanoparticles is reviewed with a focus on the chemical properties of the utilized polymers. © 2017 Society of Chemical Industry     

叶酸受体介导的CdS纳米颗粒的生物成像

以巯基乙酸为修饰剂,合成了具有特殊光学性质的水溶性硫化镉( CdS)纳米颗粒.通过红外吸收光谱、荧光光谱和透射电子显微镜对产物的光学性能和晶体结构进行了表征,结果表明经巯基乙酸改性、聚乙烯亚胺及叶酸修饰的CdS纳米颗粒为球形,粒径约180 nm左右,并且有较高产率,同时具有良好的分散性、稳定性及生物相容性;利用激光扫描...     

Polymers for siRNA delivery: inspired by viruses to be targeted, dynamic, and precise

Synthetic small interfering RNA (siRNA) presents an exciting novel medical opportunity. Although researchers agree that siRNA could have a great therapeutic impact, the required extracellular and intracellular delivery of these molecules into the disease-associated target cells presents the primary roadblock for the broader translation of these molecules into medicines. Thus, the design of adequate delivery technologies has utmost importance. Viruses are natural masterpieces of nucleic acid delivery and present chemists and drug delivery experts with a template for the design of artificial carriers for synthetic nucleic acids such as siRNA. They have been developed into gene vectors and have provided convincing successes in gene therapy. Optimized by biological evolution, viruses are programmed to be dynamic and bioresponsive as they enter living cells, and they carry out their functions in a precisely defined sequence. However, because they are synthesized within living cells and with naturally available nucleotides and amino acids, the chemistry of viruses is limited. With the use of diverse synthetic molecules and macromolecules, chemists can provide delivery solutions beyond the scope of the natural evolution of viruses. This Account describes the design and synthesis of "synthetic siRNA viruses." These structures contain elements that mimic the delivery functions of viral particles and surface domains that shield against undesired biological interactions and enable specific host cell receptor binding through the presentation of multiple targeting ligands. For example, cationic polymers can reversibly package one or more siRNA molecules into nanoparticle cores to protect them against a degradative bioenvironment. After internalization by receptor-mediated endocytosis into the acidifying endosomes of cells, synthetic siRNA can escape from these vesicles through the activation of membrane-disruption domains as viruses do and reach the cytoplasm, the location of RNA interference. This multistep task presents an attractive challenge for chemists. Similar to the design of prodrugs, the functional domains of these systems have to be activated in a dynamic mode, triggered by conformational changes or bond cleavages in the relevant microenvironment such as the acidic endosome or disulfide-reducing cytoplasm. These chemical analogues of viral domains are often synthetically simpler and more easily accessible molecules than viral proteins. Their precise assembly into multifunctional macromolecular and supramolecular structures is facilitated by improved analytical techniques, precise orthogonal conjugation chemistries, and sequence-defined polymer syntheses. The chemical evolution of microdomains using chemical libraries and macromolecular and supramolecular evolution could provide key strategies for optimizing siRNA carriers to selected medical indications.     

Bioorthogonal chemistry based on-demand drug delivery system in cancer therapy

Benefiting from the advantage of taking place in biological environments without interfering with an innate biochemical process, the bioorthogonal reaction that commonly contains the “bond formation” and “bond cleavage” system has been widely used in targeted therapy for a variety of tumors. Herein, several prominent cases based on the bioorthogonal reaction that tailoring the metabolic glycoengineering tactics to modified cells for cancer immunotherapy, and the innovative tactics for reducing the metal ions’ toxic and side effects with microneedle patches will be highlighted. Based on these applications, the complexities, potential pitfalls, and opportunities of bioorthogonal chemistry in future cancer therapy will be evaluated.     

Polymeric siRNA delivery vectors: knocking down cancers with polymeric‐based gene delivery systems

One of the most promising routes for cancer therapy that has evolved over the previous decade is the use of small‐interfering RNA (siRNA) as a means of switching off genes that are responsible for tumour development. However, while siRNA and gene/antisense therapies provide alternatives to conventional chemotherapies, significant hurdles related to the delivery and efficacy of treatment must still be overcome before this technology can be used as an effective treatment for cancer and other diseases. This review highlights the issues associated with siRNA therapy in vivo, and describes the various approaches that are being explored using polymers as delivery vectors. In particular, the review focuses on targeted delivery as a means of improving efficacy of the gene therapy. © 2014 Society of Chemical Industry     

In vivo targeted delivery of nanoparticles for theranosis

Therapy and diagnosis are two major categories in the clinical treatment of disease. Recently, the word "theranosis" has been created, combining the words to describe the implementation of these two distinct pursuits simultaneously. For successful theranosis, the efficient delivery of imaging agents and drugs is critical to provide sufficient imaging signal or drug concentration in the targeted disease site. To achieve this purpose, biomedical researchers have developed various nanoparticles composed of organic or inorganic materials. However, the targeted delivery of these nanoparticles in animal models and patients remains a difficult hurdle for many researchers, even if they show useful properties in cell culture condition. In this Account, we review our strategies for developing theranostic nanoparticles to accomplish in vivo targeted delivery of imaging agents and drugs. By applying these rational strategies, we achieved fine multimodal imaging and successful therapy. Our first strategy involves physicochemical optimization of nanoparticles for long circulation and an enhanced permeation and retention (EPR) effect. We accomplished this result by testing various materials in mouse models and optimizing the physical properties of the materials with imaging techniques. Through these experiments, we developed a glycol chitosan nanoparticle (CNP), which is suitable for angiogenic diseases, such as cancers, even without an additional targeting moiety. The in vivo mechanism of this particle was examined through rationally designed experiments. In addition, we evaluated and compared the biodistribution and target-site accumulation of bare and drug-loaded nanoparticles. We then focus on the targeting moieties that bind to cell surface receptors. Small peptides were selected as targeting moieties because of their stability, low cost, size, and activity per unit mass. Through phage display screening, the interleukin-4 receptor binding peptide was discovered, and we combined it with our nanoparticles. This product accumulated efficiently in atherosclerotic regions or tumors during both imaging and therapy. We also developed hyaluronic acid nanoparticles that can bind efficiently to the CD44 antigen receptors abundant in many tumor cells. Their delivery mechanism is based on both physicochemical optimization for the EPR effect and receptor-mediated endocytosis by their hyaluronic acid backbone. Finally, we introduce the stimuli-responsive system related to the chemical and biological changes in the target disease site. Considering the relatively low pH in tumors and ischemic sites, we applied pH-sensitive micelle to optical imaging, magnetic resonance imaging, anticancer drug delivery, and photodynamic therapy. In addition, we successfully evaluated the in vivo imaging of enzyme activity at the target site with an enzyme-specific peptide sequence and CNPs. On the basis of these strategies, we were able to develop self-assembled nanoparticles for in vivo targeted delivery, and successful results were obtained with them in animal models for both imaging and therapy. We anticipate that these rational strategies, as well as our nanoparticles, will be applied in both the diagnosis and therapy of many human diseases. These theranostic nanoparticles are expected to greatly contribute to optimized therapy for individual patients as personalized medicine, in the near future.     

Development of viral nanoparticles for efficient intracellular delivery

Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5.     

Inorganic nanoparticles as carriers for efficient cellular delivery

Cellular delivery involving the transfer of various drugs and bio-active molecules (peptides, proteins and DNAs, etc.) through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. This topic has been extensively reviewed. The direct delivery of drugs and biomolecules, however, is generally inefficient and suffering from problems such as enzymic degradation of DNAs. Therefore, searching for efficient and safe transport vehicles (carriers) to delivery genes or drugs into cells has been challenging yet exciting area of research. In past decades, many carriers have been developed and investigated extensively which can be generally classified into four major groups: viral carriers, organic cationic compounds, recombinant protiens and inorganic nanoparticles. Many inorganic materials, such as calcium phosphate, gold, carbon materials, silicon oxide, iron oxide and layered double hydroxide (LDH), have been studied. Inorganic nanoparticles show low toxicity and promise for controlled delivery properties, thus presenting a new alternative to viral carriers and cationic carriers. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal tissues) and controlled release of carried drugs. This paper reviews the latest advances in inorganic nanoparticle applications as cellular delivery carriers and highlights some key issues in efficient cellular delivery using inorganic nanoparticles. Critical properties of inorganic nanoparticles, surface functionalisation (modification), uptake of biomolecules, the driving forces for delivery, and release of biomolecules will be reviewed systematically. Selected examples of promising inorganic nanoparticle delivery systems, including gold, fullerences and carbon nanotubes, LDH and various oxide nanoparticles in particular their applications for gene delivery will be discussed. The fundamental understanding of properties of inorganic nanoparticles in relation to cellular delivery efficiency as the most paramount issue will be highlighted.