A Nanobody‐Conjugated DNA Nanoplatform for Targeted Platinum‐Drug Delivery

The targeted delivery of chemotherapeutic drugs is a major challenge in the clinical treatment of cancer. Herein, we constructed a multifunctional DNA nanoplatform as a versatile carrier of the highly potent platinum‐based DNA intercalator, 56MESS. In our rational design, 56MESS was efficiently loaded into the double‐bundle DNA tetrahedron through intercalation with the DNA duplex. With the integration of a nanobody that both targets and blocks epidermal growth factor receptor (EGFR), the DNA nanocarriers exhibit excellent selectivity for cells with elevated EGFR expression (a common biomarker related to tumor formation) and combined tumor therapy without obvious systemic toxicity. This DNA‐based platinum‐drug delivery system provides a promising strategy for the treatment of tumors.     

Enzymatic Prenylation and Oxime Ligation for the Synthesis of Stable and Homogeneous Protein–Drug Conjugates for Targeted Therapy

Targeted therapy based on protein–drug conjugates has attracted significant attention owing to its high efficacy and low side effects. However, efficient and stable drug conjugation to a protein binder remains a challenge. Herein, a chemoenzymatic method to generate highly stable and homogenous drug conjugates with high efficiency is presented. The approach comprises the insertion of the CaaX sequence at the C‐terminal end of the protein binder, prenylation using farnesyltransferase, and drug conjugation through an oxime ligation reaction. MMAF and an EGFR‐specific repebody are used as the antitumor agent and protein binder, respectively. The method enables the precisely controlled synthesis of repebody–drug conjugates with high yield and homogeneity. The utility of this approach is illustrated by the notable stability of the repebody–drug conjugates in human plasma, negligible off‐target effects, and a remarkable antitumor activity in vivo. The present method can be widely used for generating highly homogeneous and stable PDCs for targeted therapy.     

Self-Assembled Plasmonic Vesicles of SERS-Encoded Amphiphilic Gold Nanoparticles for Cancer Cell Targeting and Traceable Intracellular Drug Delivery

We report the development of bioconjugated plasmonic vesicles assembled from SERS-encoded amphiphilic gold nanoparticles for cancer-targeted drug delivery. This new type of plasmonic assemblies with a hollow cavity can play multifunctional roles as delivery carriers for anticancer drugs and SERS-active plasmonic imaging probes to specifically label targeted cancer cells and monitor intracellular drug delivery. We have shown that the pH-responsive disassembly of the plasmonic vesicle, stimulated by the hydrophobic-to-hydrophilic transition of the hydrophobic brushes in acidic intracellular compartments, allows for triggered intracellular drug release. Because self-assembled plasmonic vesicles exhibit significantly different plasmonic properties and greatly enhanced SERS intensity in comparison with single gold nanoparticles due to strong interparticle plasmonic coupling, disassembly of the vesicles in endocytic compartments leads to dramatic changes in scattering properties and SERS signals, which can serve as independent feedback mechanisms to signal cargo release from the vesicles. The unique structural and optical properties of the plasmonic vesicle have made it a promising platform for targeted combination therapy and theranostic applications by taking advantage of recent advances in gold nanostructure based in vivo bioimaging and photothermal therapy and their loading capacity for both hydrophilic (nucleic acids and proteins) and hydrophobic (small molecules) therapeutic agents.     

A new A431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A₄₃₁ cells) are an important target for drug discovery. We have developed a new A₄₃₁/cell membrane chromatography (A₄₃₁/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A₄₃₁ cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A₄₃₁/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A₄₃₁ cell growth in vitro. This new A₄₃₁/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

Therapeutic Potential of DNAzyme Loaded on Chitosan/Cyclodextrin Nanoparticle to Recovery of Chemosensitivity in the MCF-7 Cell Line

Commonly, acquired resistances to anticancer drug are mediated by overexpression of a membrane-associated protein that encode via multi-drug resistance gene-1 (MDR1). Herein, the mRNA-cleaving DNAzyme that targets the mRNA of MDR1 gene in doxorubicin-resistant breast cancer cell line (MCF-7/DR) loaded on the chitosan β-cyclodextrin complexes was used as a tropical agent. Chitosan/β-cyclodextrin complexes were used to deliver DNAzymes into cancer cells. Determination of the physicochemical characteristics of the particles was done by photon correlation spectroscopy and scanning electron microscopy. The encapsulation efficiency of the complexes was tested by using gel retardation assay. Positively charged nanoparticles interacted with DNAzyme that could perform as an efficient DNAzyme transfection system. The rationale usage of this platform is to sensitize MCF-7/DR to doxorubicin by downregulating the drug-resistance gene MDR1. Results demonstrated a downregulation of MDR1 mRNAs in MCF-7/DR/DNZ by real-time PCR, compared to the MCF-7/DR as control. WST1 assay showed the 22-fold decrease in drug resistance on treated cells 24 h after transfection. Results showed the intracellular accumulation of Rh123 increased in the treated cells with DNAzyme. Results suggested a potential platform in association with chemotherapy drug for cancer therapy and indicated extremely efficient at delivery of DNAzyme in restoring chemosensitivity.

     

Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

Nanobodies can be seen as next‐generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site‐specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.     

Four‐fold Channel‐Nicked Human Ferritin Nanocages for Active Drug Loading and pH‐Responsive Drug Release

Human ferritins are emerging platforms for non‐toxic protein‐based drug delivery, owing to their intrinsic or acquirable targeting abilities to cancer cells and hollow cage structures for drug loading. However, reliable strategies for high‐level drug encapsulation within ferritin cavities and prompt cellular drug release are still lacking. Ferritin nanocages were developed with partially opened hydrophobic channels, which provide stable routes for spontaneous and highly accumulated loading of Fe^II‐conjugated drugs as well as pH‐responsive rapid drug release at endoplasmic pH. Multiple cancer‐related compounds, such as doxorubicin, curcumin, and quercetin, were actively and heavily loaded onto the prepared nicked ferritin. Drugs on these minimally modified ferritins were effectively delivered inside cancer cells with high toxicity.     

Mutant‐Selective Allosteric EGFR Degraders are Effective Against a Broad Range of Drug‐Resistant Mutations

Targeting epidermal growth factor receptor (EGFR) through an allosteric mechanism provides a potential therapeutic strategy to overcome drug‐resistant EGFR mutations that emerge within the ATP binding site. Here, we develop an allosteric EGFR degrader, DDC‐01‐163, which can selectively inhibit the proliferation of L858R/T790M (L/T) mutant Ba/F3 cells while leaving wildtype EGFR Ba/F3 cells unaffected. DDC‐01‐163 is also effective against osimertinib‐resistant cells with L/T/C797S and L/T/L718Q EGFR mutations. When combined with an ATP‐site EGFR inhibitor, osimertinib, the anti‐proliferative activity of DDC‐01‐163 against L858R/T790M EGFR‐Ba/F3 cells is enhanced. Collectively, DDC‐01‐163 is a promising allosteric EGFR degrader with selective activity against various clinically relevant EGFR mutants as a single agent and when combined with an ATP‐site inhibitor. Our data suggests that targeted protein degradation is a promising drug development approach for mutant EGFR.     

Antibody modified Bovine Serum Albumin microspheres for targeted delivery of anticancer agent Gemcitabine

Inhibition of the EGFR signaling pathway is one of the attractive therapeutic targets for pancreatic cancer as recent studies demonstrated that EGFR is over‐expressed in pancreatic cancer. In this article we have demonstrated the design of targeted drug delivery system containing Bovine Serum Albumin (BSA) microspheres as delivery vehicle, gemcitabine as anticancer drug and anti‐EGFR (epidermal growth factor receptor) monoclonal antibody as targeting agent. The conjugated BSA microspheres were characterized by several physico‐chemical techniques such as scanning electron microscope, optical microscopy, fluorescent microscopy etc. Administration of these BSA microspheres containing gemcitabine and anti‐EGFR (BSA‐Gem‐EGFR) shows significant inhibition of pancreatic cancer cells (AsPC1) compared to the cells treated with only BSA microspheres, BSA with gemcitabine (BSA‐Gem), and free gemcitabine. This strategy could be used as a generalized approach for the treatment of pancreatic cancer along with other cancers which overexpress EGFR on cell surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery

The development of targeted vehicles for systemic drug delivery relies on optimizing both the cell-targeting ligand and the physicochemical characteristics of the nanoparticle carrier. A versatile platform based on modification of gold nanoparticles with thiolated polymers is presented in which design parameters can be varied independently and systematically. Nanoparticle formulations of varying particle size, surface charge, surface hydrophilicity, and galactose ligand density were prepared by conjugation of PEG-thiol and galactose-PEG-thiol to gold colloids. This platform was applied to screen for nanoparticle formulations that demonstrate hepatocyte-targeted delivery in vivo. Nanoparticle size and the presence of galactose ligands were found to significantly impact the targeting efficiency. Thus, this platform can be readily applied to determine design parameters for targeted drug delivery systems.Modified gold nanoparticles are a suitable model for nanoparticle-based gene carriers.     

Doxorubicin Encapsulated in TPGS-Modified 2D-Nanodisks Overcomes Multidrug Resistance

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d -α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.     

Gelatin Coating for the Improvement of Stability and Cell Uptake of Hydrophobic Drug-Containing Liposomes

Purpose: Most therapeutic agents have limitations owing to low selectivity and poor solubility, resulting in post-treatment side effects. Therefore, there is a need to improve solubility and develop new formulations to deliver therapeutic agents specifically to the target site. Gelatin is a natural protein that is composed of several amino acids. Previous studies revealed that gelatin contains arginyl-glycyl-aspartic acid (RGD) sequences that become ligands for the integrin receptors expressed on cancer cells. Thus, in this study, we aimed to increase the efficiency of drug delivery into cancer cells by coating drug-encapsulating liposomes with gelatin (gelatin-coated liposomes, GCLs). Methods: Liposomes were coated with gelatin using electrostatic interaction and covalent bonding. GCLs were compared with PEGylated liposomes in terms of their size, zeta potential, encapsulation efficiency, stability, dissolution profile, and cell uptake. Results: Small-sized and physically stable GCLs were prepared, and they showed high drug-encapsulation efficiency. An in vitro dissolution study showed sustained release depending on the degree of gelatin coating. Cell uptake studies showed that GCLs were superior to PEGylated liposomes in terms of cancer cell-targeting ability. Conclusions: GCLs can be a novel and promising carrier system for targeted anticancer agent delivery. GCLs, which exhibited various characteristics depending on the coating degree, could be utilized in various ways in future studies.     

pH/酶/光热多重响应的金纳米笼/透明质酸核壳结构纳米载体的制备与性能

以金纳米笼(AuNC)为核, 巯基化改性的透明质酸(LC-HA)为壳, 盐酸阿霉素(DOX)为药物模型, 通过简单的一锅法制备了核壳结构载药纳米粒子DOX@AuNC@HA(DAH). 金纳米笼为药物装载提供容器且赋予载体光热性能, 改性的透明质酸对金纳米笼进行包封并提供pH/酶响应及靶向介导功能. 对DAH的结构进行了表征, 并进行了载药、 控释性能以及细胞摄取和细胞毒性的研究. 结果表明, 核壳结构纳米微粒DAH具有较高的载药能力, 在激光源的照射下具有较好的循环稳定性和较高的光热转换率. 在pH=7.4的磷酸盐缓冲液中, DAH具有较高的稳定性, 20 h的药物泄露率低于20%; 而在酸性环境、 透明质酸酶(HAase)及光热作用下, DAH均能较快地释放出装载的药物, 展现出较好的刺激响应性. 此外, DAH能够更多地被肿瘤细胞摄取, 表现出一定的靶向性; 当化疗与光热疗法共同作用时, 肿瘤细胞的活性大大减弱, 展现出了联合疗法的优势及潜力.     

Photoresponsive DNA-cross-linked hydrogels for controllable release and cancer therapy

We have developed a photoresponsive DNA-cross-linked hydrogel that can be photoregulated by two wavelengths with a reversible sol-gel conversion. This photoinduced conversion can be further utilized for precisely controllable encapsulation and release of multiple loads. Specifically, photosensitive azobenzene moieties are incorporated into DNA strands as cross-linkers, such that their hybridization to complementary DNAs (cDNAs) responds differently to different wavelengths of light. On the basis of the rheology variation of hydrogels, it is possible to utilize this material for storing and releasing molecules and nanoparticles. To prove the concept, three different materials--fluorescein, horseradish peroxidase, and gold nanoparticles--were encapsulated inside the gel at 450 nm and then released by photons at 350 nm. Further experiments were carried out to deliver the chemotherapy drug doxorubicin in a similar manner in vitro. Our results show a net release rate of 65% within 10 min, and the released drug maintained its therapeutic effect. This hydrogel system provides a promising platform for drug delivery in targeted therapy and in biotechnological applications.     

Photobodies: Light-Activatable Single-Domain Antibody Fragments

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen-binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single-domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o-Nitrobenzyl-protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti-GFP photobody exhibited an at least 10 000-fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.     

A simple synthesis of a targeted drug delivery system with enhanced cytotoxicity

A simple synthesis of a targeted drug delivery system with enhanced cytotoxicity to (epidermal growth factor receptor) EGFR(+) cancer cells.     

Chitosan-Linseed mucilage polyelectrolyte complex nanoparticles of Methotrexate: In vitro cytotoxic efficacy and toxicological studies

The goal of this research was to develop, fabricate and analyze polymeric nanoparticles for the administration of methotrexate (MTX). Linseed mucilage and chitosan nanoparticles (NPs) were prepared using a slightly modified polyelectrolyte complex (PEC) method. The size, shape, and encapsulation effectiveness of the resultant nanoparticles were measured. MTX release profiles at gastrointestinal pH (1.2 and 7.4) and tumor pH (5.5) were examined to determine the targeted potential of NPs as pH-responsive nanocarriers. Zeta analysis showed that nanoparticles prepared by PEC have a size range of 192.1 nm to 246 nm, and PDI was 0.3 of the optimized formulation, which showed homogenous nature of prepared nanoparticles formulation. The findings demonstrated that NPs have a low polydispersity index and a positive zeta potential (PDI). The in-vitro release of the drug indicated a pH-dependent, sustained drug release up to 24 h. Blank LSMCSNPs had almost no in-vivo cytotoxicity for 14 days, while optimum MTX loaded NPs had strong antitumor effects on HepG2 and MCF-7 cells as measured by the MTT assay. Cell apoptosis induction was also checked and MCF-7 cells treated with MTX-LSMCSNPs had a significantly greater rate of apoptosis (21.2 %) than those treated with MTX alone (14.14 %). The findings show that LSMCSNPs could be a potential delivery mechanism for methotrexate to cancer cells in a secure, steady, and ideally controlled manner to improve therapeutic outcomes.     

用高表达EGFR细胞膜色谱-HPLC/MS联用快速筛选独活中抗EGFR活性成分

报道了用高表达表皮生长因子受体细胞膜色谱与高效液相色谱/质谱在线联用方法(EGFR/CMC-online-HPLC/MS)快速筛选发现中药独活中的活性成分.实验中,采用高表达EGFR的细胞膜制备色谱固定相,建立EGFR/细胞膜色谱(EGFR/CMC)模型,利用柱切换和固相萃取技术,将EGFR/CMC模型与高效液相色谱/质谱(HPLC/MS)在线联用,构成一种新的可同时"识别-鉴定"目标成分的二维色谱系统,并应用于快速筛选独活中具有抗EGFR活性的目标成分.结果发现独活中的蛇床子素具有与对照药物达沙替尼类似的色谱保留特性,能够作用于EGFR;同时MTT及Elisa分析实验证实蛇床子素对HEK293EGFR细胞增殖及EGFR表达均有抑制作用.本文建立的EGFR/CMC-online-HPLC/MS二维色谱方法,可以选择性地从中药复杂体系中快速"识别-鉴定"目标组分,且筛选结果与特定生物效应显著相关.     

Targeted pH-responsive polyion complex micelle for controlled intracellular drug delivery

Cancer therapy with nanoscale drug formulations has made significant progress in the past few decades. However, the selective accumulation and release of therapeutic agents in the lesion sites are still great challenges. To this end, we developed a cRGD-decorated pH-responsive polyion complex (PIC) micelle for intracellular targeted delivery of doxorubicin (DOX) to upregulate tumor inhibition and reduce toxicity. The PIC micelle was self-assembled via the electrostatic interaction between the positively charged cRGD-modified poly(ethylene glycol)-block-poly(l-lysine) and the anionic acid-sensitive 2,3-dimethylmaleic anhydride-modified doxorubicin (DAD). The decoration of cRGD enhanced the cell internalization of PIC micelle through the specific recognition of α_vβ₃ integrin on the membrane of tumor cells. The active DOX was released under intracellular acidic microenvironment after endocytosis following the decomposition of DAD. Moreover, the targeted PIC micelle exhibited enhanced inhibition efficacies toward hepatoma in vitro and in vivo compared with the insensitive controls. The smart multifunctional micelle provides a promising platform for target intracellular delivery of therapeutic agent in cancer therapy.