A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.     

An iGlu Receptor Antagonist and Its Simultaneous Use with an Anticancer Drug for Cancer Therapy

Glutamate receptor antagonists have been known to play a crucial role in the treatment of many neuronal diseases. Recently, these antagonists have also shown therapeutic effects in the treatment of cancer. In this study, an ionotropic glutamate (iGlu) receptor antagonist, 4‐hydroxyphenylacetyl spermine ( L1 ), was used concurrently with a common anticancer drug, doxorubicin (Dox), for simultaneous cancer therapy. Mesoporous silica nanoparticles (MSNPs) were employed as the delivery vehicle for both L1 and Dox by conjugating the iGlu receptor antagonist on the surface and encapsulating Dox within the mesopores. Dox was then trapped within the mesopores by functionalizing a redox‐cleavable capping group on the MSNP surface, and it could be released upon exposure to the reductive glutathione. In vitro studies on B16F10 and NIH3T3 cell lines revealed that the iGlu receptor antagonist L1 exhibited therapeutic as well as targeting effects. In addition, the simultaneous use of therapeutic L1 and Dox proved to be synergistic in the treatment of cancer. The present work demonstrated the feasibility of employing a delivery system to deliver both neuroprotective drug and anticancer drug for efficient anticancer treatment.     

Albumin-Albumin/Lactosylated Core-Shell Nanoparticles: Therapy to Treat Hepatocellular Carcinoma for Controlled Delivery of Doxorubicin

Doxorubicin (Dox) is the most widely used chemotherapeutic agent and is considered a highly powerful and broad-spectrum for cancer treatment. However, its application is compromised by the cumulative side effect of dose-dependent cardiotoxicity. Because of this, targeted drug delivery systems (DDS) are currently being explored in an attempt to reduce Dox systemic side-effects. In this study, DDS targeting hepatocellular carcinoma (HCC) has been designed, specifically to the asialoglycoprotein receptor (ASGPR). Dox-loaded albumin-albumin/lactosylated (core-shell) nanoparticles (tBSA/BSALac NPs) with low (LC) and high (HC) crosslink using glutaraldehyde were synthesized. Nanoparticles presented spherical shapes with a size distribution of 257 ± 14 nm and 254 ± 14 nm, as well as an estimated surface charge of −28.0 ± 0.1 mV and −26.0 ± 0.2 mV, respectively. The encapsulation efficiency of Dox for the two types of nanoparticles was higher than 80%. The in vitro drug release results showed a sustained and controlled release profile. Additionally, the nanoparticles were revealed to be biocompatible with red blood cells (RBCs) and human liver cancer cells (HepG2 cells). In cytotoxicity assays, Dox-loaded nanoparticles decrease cell viability more efficiently than free Dox. Specific biorecognition assays confirmed the interaction between nanoparticles and HepG2 cells, especially with ASGPRs. Both types of nanoparticles may be possible DDS specifically targeting HCC, thus reducing side effects, mainly cardiotoxicity. Therefore, improving the quality of life from patients during chemotherapy.     

Self‐Assembled Aptamer‐Based Drug Carriers for Bispecific Cytotoxicity to Cancer Cells

Monovalent aptamers can deliver drugs to target cells by specific recognition. However, different cancer subtypes are distinguished by heterogeneous biomarkers and one single aptamer is unable to recognize all clinical samples from different patients with even the same type of cancers. To address heterogeneity among cancer subtypes for targeted drug delivery, as a model, we developed a drug carrier with a broader recognition range of cancer subtypes. This carrier, sgc8c‐sgd5a (SD), was self‐assembled from two modified monovalent aptamers. It showed bispecific recognition abilities to target cells in cell mixtures; thus broadening the recognition capabilities of its parent aptamers. The self‐assembly of SD simultaneously formed multiple drug loading sites for the anticancer drug doxorubicin (Dox). The Dox‐loaded SD (SD–Dox) also showed bispecific abilities for target cell binding and drug delivery. Most importantly, SD–Dox induced bispecific cytotoxicity in target cells in cell mixtures. Therefore, by broadening the otherwise limited recognition capabilities of monovalent aptamers, bispecific aptamer‐based drug carriers would facilitate aptamer applications for clinically heterogeneous cancer subtypes that respond to the same cancer therapy.     

Chemical Design of Nuclear‐Targeting Mesoporous Silica Nanoparticles for Intra‐nuclear Drug Delivery

Targeted drug delivery has been widely explored for efficient tumor therapy with desired efficacy but minimized side effects. It is widely known that large numbers of DNA‐toxins, such as doxorubicin, genes, reactive oxygen species, serving as therapeutic agents, can result in maximized therapeutic effects via the interaction directly with DNA helix. So after cellular uptake, these agents should be further delivered into cell nuclei to play their essential roles in damaging the DNA helix in cancer cells. Here, we demonstrate the first paradigm established in our laboratory in developing nuclear‐targeted drug delivery systems (DDSs) based on MSNs for enhanced therapeutic efficiency in the hope of speeding their translation into the clinics. Firstly, nuclear‐targeting DDSs based on MSNs, capable of intranuclear accumulation and drug release therein, were designed and constructed for the first time, resulting in much enhanced anticancer effects both in vitro and in vivo. Such an MSNs‐based and nuclear‐targeted drug/agent delivery strategy was further applied to overcome multidrug resistance (MDR) of malignant tumors, intra‐nuclearly deliver therapeutic genes, photosensitizers, radio‐enhancement agents and photothermal agents to realize efficient gene therapy, photodynamic therapy, radiation therapy and photothermal therapy, respectively.     

上转换纳米粒子结合DNA链式扩增技术在阿霉素光控释放中的应用

纳米技术的发展使得纳米材料可以通过不同的表面包覆和修饰而在生物医药中发挥应用。 构建简单、经济、药物释放可控的生物相容性纳米药物仍是纳米生物化学领域的重点。 我们构建的纳米载药体系(DDS)以NaYF₄：Yb/Tm上转换纳米粒子为载体,在其表面通过光致断键型小分子4,5-二甲氧基-2-硝基苯基乙酮(DMNPE)连接一段短单链DNA,利用DNA链式扩增技术(HCR)来调节纳米粒子最终修饰的双链DNA的总量,从而控制对抗癌药物阿霉素(Dox)的担载量,在980 nm激光照射下上转换纳米粒子发射可切断DMNPE连接的近紫外光,协同胞内DNA酶的作用达到对药物的可控释放。 由于近红外光照对生物组织具有较好的穿透能力,此体系能够对病灶位置有更好的光靶向性从而减少药物的毒副作用。     

Integrated Hollow Mesoporous Silica Nanoparticles for Target Drug/siRNA Co‐Delivery

A hollow mesoporous silica nanoparticle (HMSNP) based drug/siRNA co‐delivery system was designed and fabricated, aiming at overcoming multidrug resistance (MDR) in cancer cells for targeted cancer therapy. The as‐prepared HMSNPs have perpendicular nanochannels connecting to the internal hollow cores, thereby facilitating drug loading and release. The extra volume of the hollow core enhances the drug loading capacity by two folds as compared with conventional mesoporous silica nanoparticles (MSNPs). Folic acid conjugated polyethyleneimine (PEI‐FA) was coated on the HMSNP surfaces under neutral conditions through electrostatic interactions between the partially charged amino groups of PEI‐FA and the phosphate groups on the HMSNP surfaces, blocking the mesopores and preventing the loaded drugs from leakage. Folic acid acts as the targeting ligand that enables the co‐delivery system to selectively bind with and enter into the target cancer cells. PEI‐FA‐coated HMSNPs show enhanced siRNA binding capability on account of electrostatic interactions between the amino groups of PEI‐FA and siRNA, as compared with that of MSNPs. The electrostatic interactions provide the feasibility of pH‐controlled release. In vitro pH‐responsive drug/siRNA co‐delivery experiments were conducted on HeLa cell lines with high folic acid receptor expression and MCF‐7 cell lines with low folic acid receptor expression for comparison, showing effective target delivery to the HeLa cells through folic acid receptor meditated cellular endocytosis. The pH‐responsive intracellular drug/siRNA release greatly minimizes the prerelease and possible side effects of the delivery system. By simultaneously delivering both doxorubicin (Dox) and siRNA against the Bcl‐2 protein into the HeLa cells, the expression of the anti‐apoptotic protein Bcl‐2 was successfully suppressed, leading to an enhanced therapeutic efficacy. Thus, the present multifunctional nanoparticles show promising potentials for controlled and targeted drug and gene co‐delivery in cancer treatment.     

Factors Affecting Extracellular Vesicles Based Drug Delivery Systems

Extracellular vesicles (EVs) play major roles in intracellular communication and participate in several biological functions in both normal and pathological conditions. Surface modification of EVs via various ligands, such as proteins, peptides, or aptamers, offers great potential as a means to achieve targeted delivery of therapeutic cargo, i.e., in drug delivery systems (DDS). This review summarizes recent studies pertaining to the development of EV-based DDS and its advantages compared to conventional nano drug delivery systems (NDDS). First, we compare liposomes and exosomes in terms of their distinct benefits in DDS. Second, we analyze what to consider for achieving better isolation, yield, and characterization of EVs for DDS. Third, we summarize different methods for the modification of surface of EVs, followed by discussion about different origins of EVs and their role in developing DDS. Next, several major methods for encapsulating therapeutic cargos in EVs have been summarized. Finally, we discuss key challenges and pose important open questions which warrant further investigation to develop more effective EV-based DDS.     

DNA origami as a carrier for circumvention of drug resistance

Although a multitude of promising anti-cancer drugs have been developed over the past 50 years, effective delivery of the drugs to diseased cells remains a challenge. Recently, nanoparticles have been used as drug delivery vehicles due to their high delivery efficiencies and the possibility to circumvent cellular drug resistance. However, the lack of biocompatibility and inability to engineer spatially addressable surfaces for multi-functional activity remains an obstacle to their widespread use. Here we present a novel drug carrier system based on self-assembled, spatially addressable DNA origami nanostructures that confronts these limitations. Doxorubicin, a well-known anti-cancer drug, was non-covalently attached to DNA origami nanostructures through intercalation. A high level of drug loading efficiency was achieved, and the complex exhibited prominent cytotoxicity not only to regular human breast adenocarcinoma cancer cells (MCF?7), but more importantly to doxorubicin-resistant cancer cells, inducing a remarkable reversal of phenotype resistance. With the DNA origami drug delivery vehicles, the cellular internalization of doxorubicin was increased, which contributed to the significant enhancement of cell-killing activity to doxorubicin-resistant MCF?7 cells. Presumably, the activity of doxorubicin-loaded DNA origami inhibits lysosomal acidification, resulting in cellular redistribution of the drug to action sites. Our results suggest that DNA origami has immense potential as an efficient, biocompatible drug carrier and delivery vehicle in the treatment of cancer.     

Near‐Infrared‐Controlled,Targeted Hydrophobic Drug‐Delivery System for Synergistic Cancer Therapy

Hydrophobicity has been an obstacle that hinders the use of many anticancer drugs. A critical challenge for cancer therapy concerns the limited availability of effective biocompatible delivery systems for most hydrophobic therapeutic anticancer drugs. In this study, we have developed a targeted near‐infrared (NIR)‐regulated hydrophobic drug‐delivery platform based on gold nanorods incorporated within a mesoporous silica framework (AuMPs). Upon application of NIR light, the photothermal effect of the gold nanorods leads to a rapid rise in the local temperature, thus resulting in the release of the entrapped drug molecules. By integrating chemotherapy and photothermotherapy into one system, we have studied the therapeutic effects of camptothecin‐loaded AuMP‐polyethylene glycol‐folic acid nanocarrier. Results revealed a synergistic effect in vitro and in vivo, which would make it possible to enhance the therapeutic effect of hydrophobic drugs and decrease drug side effects. Studies have shown the feasibility of using this nanocarrier as a targeted and noninvasive remote‐controlled hydrophobic drug‐delivery system with high spatial/temperal resolution. Owing to these advantages, we envision that this NIR‐controlled, targeted drug‐delivery method would promote the development of high‐performance hydrophobic anticancer drug‐delivery system in future clinical applications.     

A microRNA-21-responsive doxorubicin-releasing sticky-flare for synergistic anticancer with silencing of microRNA and chemotherapy

A sticky-flare gold nanoparticle probe(AuNP-probe) is designed by the combination of locked nucleic acid functionalized silencing of microRNA technology for intracellular microRNA-21(miRNA-21) sensitively detecting, fluorescence imaging,localizing and silencing. The limit of detection is as low as 0.01 n M. Overexpressed miRNA-21 in cancer cells serves as endogenous drug release stimuli to trigger the release of probe-loaded doxorubicin(Dox), which soon translocates into cell nuclei. This multifunctional Dox-loaded AuNP-probe(Dox-AuNP-probe) could induce cancer cell apoptosis effectively through the synergistic effect of gene silencing and chemotherapy. This Dox-AuNP-probe exhibits superior drug potency compared to free Dox molecules, with a cell inhibition rate of 57%(but only 20% for Dox) to wild-type cancer cells and 30%(but 0% for Dox) to drug-resistent cancer cells after 72 h, and this strategy not only has the function of sensing, but also can effectively bypass drug resistance. In MCF-7 xenograft tumor-bearing mice, the Dox-AuNP-probes show greater inhibition for tumor tissues than miRNA-21 targeted AuNP-probes(Targeting-AuNP-probe) or free Dox molecules. Therefore, the Dox-AuNP-probe represents a promising nanotheranostic platform for future applications in cancer molecular imaging and therapy, especially providing a potential strategy to treat resistant cancers.     

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.     

可控自组装DNA折纸结构作为药物载体的研究进展

在过去的几十年里, DNA纳米技术作为一种快速发展的可控自组装技术, 使人们能构建出各种复杂的纳米结构. DNA折纸结构具备可编程性、 空间可寻址性、 易修饰性及良好的生物相容性等多种优越的特性, 这些优异的性质使其在药物递送方面具有广阔的应用前景. 本文总结了近年来可控自组装DNA折纸结构作为药物递送系统的研究进展, 展望了DNA折纸纳米载体未来的发展方向, 并讨论了该领域面临的挑战和可能的解决方法.     

核酸适配体在肿瘤靶向治疗方面的研究进展

传统的抗肿瘤药物大多不具有选择性,在临床治疗中产生了严重的毒副作用。核酸适配体是一种小分子核酸,能够与靶标高亲和性、高特异性地结合。选择与癌症发生发展过程密切相关的生物标记物为靶标进行SELEX过程筛选出的核酸适配体自身可作为药物,也可与药物、siRNA、纳米粒等结合构成靶向给药体系,该体系能靶向作用于特定的肿瘤细胞,降低对正常细胞的毒性,用药量显著降低,药效提高。本文综述了近年来核酸适配体直接作为抗肿瘤药物、药物载体、siRNA载体以及作为纳米材料靶向剂构成多元复合靶向给药体系在肿瘤靶向治疗领域的研究进展。     

Tumour‐Targeted Drug Delivery with Mannose‐Functionalized Nanoparticles Self‐Assembled from Amphiphilic β‐Cyclodextrins

Multivalent mannose‐functionalized nanoparticles self‐assembled from amphiphilic β‐cyclodextrins (β‐CDs) facilitate the targeted delivery of anticancer drugs to specific cancer cells. Doxorubicin (DOX)‐loaded nanoparticles equipped with multivalent mannose target units were efficiently taken up via receptor‐mediated endocytosis by MDA‐MB‐231 breast cancer cells that overexpress the mannose receptor. Upon entering the cell, the intracellular pH causes the release of DOX, which triggers apoptosis. Targeting by multivalent mannose significantly improved the capability of DOX‐loaded nanoparticles to inhibit the growth of MDA‐MB‐231 cancer cells with minimal side effects in vivo. This targeted and controlled drug delivery system holds promise as a nanotherapeutic for cancer treatment.     

Modeling of highly efficient drug delivery system induced by self-assembly of nanocarriers: A density functional study

Owing to the importance of drug delivery in cancer or other diseases’ therapy, the targeted drug delivery (TDD) system has been attracting enormous interest. Herein, we model the TDD system and design a novel rod-like nanocarrier by using the coarse grained model-based density functional theory, which combines a modified fundamental measure theory for the excluded-volume effects, Wertheim’s first-order thermodynamics perturbation theory for the chain connectivity and the mean field approximation for van der Waals attraction. For comparison, the monomer nanocarrier TDD system and the no nanocarrier one are also investigated. The results indicate that the drug delivery capacity of rod-like nanocarriers is about 62 times that of the no nanocarrier one, and about 6 times that of the monomer nanocarriers. The reason is that the rod-like nanocarriers would self-assemble into the smectic phase perpendicular to the membrane surface. It is the self-assembly of the rod-like nanocarriers that yields the driving force for the targeted delivery of drugs inside the cell membrane. By contrast, the conventional monomer nanocarrier drug delivery system lacks the driving force to deliver the drugs into the cell membrane. In short, the novel rod-like nanocarrier TDD system may improve the drug delivery efficiency. Although the model in this work is simple, it is expected that the system may provide a new perspective for cancer targeted therapy.     

Targeted therapy of SMMC-7721 liver cancer in vitro and in vivo with carbon nanotubes based drug delivery system

A new type of drug delivery system (DDS) involved chitosan (CHI) modified single walled carbon nanotubes (SWNTs) for controllable loading/release of anti-cancer doxorubicin (DOX) was constructed. CHI was non-covalently wrapped around SWNTs, imparting water-solubility and biocompatibility to the nanotubes. Folic acid (FA) was also bounded to the outer CHI layer to realize selective killing of tumor cells. The targeting DDS could effectively kill the HCC SMMC-7721 cell lines and depress the growth of liver cancer in nude mice, showing superior pharmaceutical efficiency to free DOX. The results of the blood routine and serum biochemical parameters, combined with the histological examinations of vital organs, demonstrating that the targeting DDS had negligible in vivo toxicity. Thus, this DDS is promising for high treatment efficacy and low side effects for future cancer therapy.     

Investigation of the local delivery of an intelligent chitosan-based 188Re thermosensitive in situ-forming hydrogel in an orthotopic hepatoma-bearing rat model

In this study, in vitro and in vivo evaluations of the local delivery of ¹⁸⁸Re-Tin colloid and doxorubicin (Dox) through chitosan (C)-based thermosensitive in situ-forming hydrogels by intratumoral injection in an orthotopic hepatoma-bearing rat model were carried out. Selective internal radiation therapy has been increasingly used as an alternative therapy option for hepatocellular carcinoma (HCC) and combined with biodegradable drug carrier systems to improve drug delivery and systemic toxicity. The C-based thermosensitive hydrogel (C/GP), an injectable thermogelling solution crosslinked between C and β-glycerophosphate (GP), was induced as an implanted carrier to combine the ¹⁸⁸Re-Tin colloid and Dox as a novel treatment strategy. The compounded hydrogel characteristics, including the gelation time, controlled release of Dox, and morphology, were examined. In the animal study, the biodistribution, scintigraphy, therapeutic efficacy, and histopathology were also evaluated. The characterization results reveal that C/GP/Dox hydrogels have similar gelation times of 4–4.5 min and pore sizes of as small as 10 μm compared with C/GP hydrogels. The C/GP/Dox/¹⁸⁸Re-Tin colloids have the longest release time for Dox at 2–3 days. In the in vivo experiments, both the biodistribution and scintigraphy studies have the highest hydrogel uptakes in the tumor at different time points, as well as localized radioactivities for a certain time. The therapeutic evaluation indicates that C/GP/Dox/¹⁸⁸Re-Tin colloids can more significantly inhibit tumors compared with the control group at 2 and 4 weeks post-treatment. These results indicate that this novel treatment system is a promising option for inoperable HCC.     

Advances in Targeting Drug Biological Carriers for Enhancing Tumor Therapy Efficacy

Chemotherapy drugs continue to be the main component of oncology treatment research and have been proven to be the main treatment modality in tumor therapy. However, the poor delivery efficiency of cancer therapeutic drugs and their potential off-target toxicity significantly limit their effectiveness and extensive application. The recent integration of biological carriers and functional agents is expected to camouflage synthetic biomimetic nanoparticles for targeted delivery. The promising candidates, including but not limited to red blood cells and their membranes, platelets, tumor cell membrane, bacteria, immune cell membrane, and hybrid membrane are typical representatives of biological carriers because of their excellent biocompatibility and biodegradability. Biological carriers are widely used to deliver chemotherapy drugs to improve the effectiveness of drug delivery and therapeutic efficacy in vivo, and tremendous progress is made in this field. This review summarizes recent developments in biological vectors as targeted drug delivery systems based on microenvironmental stimuli-responsive release, thus highlighting the potential applications of target drug biological carriers. The review also discusses the possibility of clinical translation, as well as the exploitation trend of these target drug biological carriers.     

核酸适体-纳米材料复合物用于癌症的诊断与靶向治疗研究进展

因具有独特的光、电、磁、热等优异性能,纳米材料已被广泛应用于生物分析与生物医学领域。核酸适体是一类能够高亲和力和高特异性地与靶标结合的寡核苷酸序列。将核酸适体作为识别单元与纳米材料相结合,可以构建核酸适体-纳米材料复合物。近年来,在肿瘤靶向治疗方面,核酸适体-纳米材料复合物受到了人们的广泛关注。通过纳米材料与具有特异性识别能力的核酸适体的结合,核酸适体-纳米材料复合物可以为癌症治疗提供一种更有效的、低毒副作用的新策略。本文综述了核酸适体-纳米材料复合物作为药物输送载体在癌症的特异性识别与诊断及靶向治疗方面的应用。除此之外,本文还总结了核酸适体-纳米材料复合物与其他新兴技术的有效结合从而提高选择性和癌症治疗效率的相关研究进展。