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.     

Metalloimmunotherapy with Rhodium and Ruthenium Complexes: Targeting Tumor-Associated Macrophages

Tumor associated macrophages (TAMs) suppress the cancer immune response and are a key target for immunotherapy. The effects of ruthenium and rhodium complexes on TAMs have not been well characterized. To address this gap in the field, a panel of 22 dirhodium and ruthenium complexes were screened against three subtypes of macrophages, triple-negative breast cancer and normal breast tissue cells. Experiments were carried out in 2D and biomimetic 3D co-culture experiments with and without irradiation with blue light. Leads were identified with cell-type-specific toxicity toward macrophage subtypes, cancer cells, or both. Experiments with 3D spheroids revealed complexes that sensitized the tumor models to the chemotherapeutic doxorubicin. Cell surface exposure of calreticulin, a known facilitator of immunogenic cell death (ICD), was increased upon treatment, along with a concomitant reduction in the M2-subtype classifier arginase. Our findings lay a strong foundation for the future development of ruthenium- and rhodium-based chemotherapies targeting TAMs.     

MicroRNA Delivery with Bioreducible Polyethylenimine as a Non‐Viral Vector for Breast Cancer Gene Therapy

Polyethylenimines (PEIs) are outstanding macromolecules belonging to the polycations used in gene transfection. The transfection efficiency and cytotoxicity of PEIs increase with the increase in their molecular weight. To break up the correlation between transfection efficiency and cytotoxicity for non‐viral gene delivery, disulfide cross‐linked polyethylenimine (PEI‐SS) has been widely employed as highly efficient gene vectors for DNA/siRNA delivery in numerous efforts. In this work, PEI‐SS is described as a non‐viral vector for miRNA delivery for the first time. PEI‐SS is synthesized via cross‐linking using disulfide bonds as the cross‐linker from low molecular weight PEI. PEI‐SS can efficiently bind anti‐miR‐155 to form the polyplex with nano‐sized spherical structures in the size range of 10–100 nm. The polyplex is degraded by glutathione (GSH, a reducing agent) in cancer cells. Anti‐miR‐155 is then released to efficiently inhibit tumor growth.     

CAGW and TAT‐NLS peptides functionalized multitargeting gene delivery system with high transfection efficiency

Gene therapy has attracted much attention in vascular tissue engineering. However, it is still challenging to develop a novel gene carrier with multifunction to overcome the barriers in gene delivery. Herein, the multitargeting gene complexes were developed based on methoxy‐poly(ethylene glycol)‐b‐poly‐(D,L‐lactide‐co‐glycolide) (mPEG‐b‐PLGA), poly(d ,l ‐lactide‐co‐glycolide)‐g‐polyethylenimine‐g‐CAGW (PLGA‐g‐PEI‐g‐CAGW), cell‐penetrating peptide YGRKKRRQRRR (TAT), nuclear localization signals (NLS), and pEGFP‐ZNF580 (pDNA) with the purpose of enhancing the transfection of endothelial cells (ECs). The low cytotoxic multitargeting gene complexes could be easily prepared by adjusting the weight ratio of mPEG‐b‐PLGA and PLGA‐g‐PEI‐g‐CAGW. Meanwhile, CAGW peptide with selectively ECs‐targeting ability and TAT‐NLS peptide sequence with both cell‐penetrating ability and nuclear targeting capacity were simultaneously introduced into gene complexes in order to enable them with the multitargeting function so as to improve their gene delivery capacity. The pDNA loading capacity of these gene complexes was confirmed by agarose gel electrophoresis assay. MTT results demonstrated that the relatively cell viability of the multitargeting gene complexes was higher than those of other groups. These multitargeting gene complexes showed higher internalization and transfection efficiencies than other groups. These results revealed that CAGW and TAT‐NLS peptide sequences benefited for efficient gene delivery. Furthermore, the wound healing assay demonstrated that the multitargeting gene complexes could promote the proliferation and migration of ECs. These results collectively demonstrated that CAGW and TAT‐NLS peptides functionalized gene delivery system could effectively enhance the transfection of ECs, which has great potential in vascular tissue engineering.     

Metronomic Photodynamic Therapy as a New Paradigm for Photodynamic Therapy: Rationale and Preclinical Evaluation of Technical Feasibility for Treating Malignant Brain Tumors¶

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low‐dose PDT using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX(PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA‐mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate, sodium salt nick‐end labeling positive) cells as compared with an acute, high‐dose regimen (ALA‐αPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectro‐fluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor‐to‐brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light‐emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.     

DNAzyme‐Loaded Metal–Organic Frameworks (MOFs) for Self‐Sufficient Gene Therapy

DNAzymes have been recognized as potent therapeutic agents for gene therapy, while their inefficient intracellular delivery and insufficient cofactor supply precludes their practical biological applications. Metal–organic frameworks (MOFs) have emerged as promising drug carriers without in‐depth consideration of their disassembled ingredients. Herein, we report a self‐sufficient MOF‐based chlorin e6‐modified DNAzyme (Ce6‐DNAzyme) therapeutic nanosystem for combined gene therapy and photodynamic therapy (PDT). The ZIF‐8 nanoparticles (NPs) could efficiently deliver the therapeutic DNAzyme without degradation into cancer cells. The pH‐responsive ZIF‐8 NPs disassemble with the concomitant release of the guest DNAzyme payloads and the host Zn²⁺ ions that serve, respectively, as messenger RNA‐targeting agent and required DNAzyme cofactors for activating gene therapy. The auxiliary photosensitizer Ce6 could produce reactive oxygen species (ROS) and provide a fluorescence signal for the imaging‐guided gene therapy/PDT.     

GRP78‐Targeted HPMA Copolymer‐Photosensitizer Conjugate for Hyperthermia‐Induced Enhanced Uptake and Cytotoxicity in MCF‐7 Breast Cancer Cells

Photodynamic therapy (PDT) is a promising cancer treatment approach. However, the photosensitizers (PS) used for PDT are often limited by their poor solubility and selectivity for tumors. The goal of this study is to improve water solubility and delivery of the photosensitizer 2‐[1‐hexyloxyethyl]‐2‐divinyl pyropheophorbide‐a (HPPH) to breast cancer cells. An N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer–HPPH photosensitizer conjugate is synthesized with heat shock receptor glucose‐regulated protein 78 (GRP78), targeting to GRP78 receptors of MCF‐7 cells, which are upregulated under mild hyperthermia. It is found that the uptake of the GRP78 targeted pep‐HPMA‐HPPH copolymer conjugate in MCF‐7 cells is improved through heat induction. Under mild hyperthermia the targeted copolymers are more effective compared to free HPPH. These results show potential for the utility of mild hyperthermia and copolymer delivery vehicles to enhance the efficacy of photodynamic therapy.     

Cyclic RGD–Polyethylene Glycol–Polyethylenimine for Intracranial Glioblastoma‐Targeted Gene Delivery

Even though the blood–brain barrier (BBB) is compromised for angiogenesis, therapeutic agents for glioblastoma multiforme (GBM) are particularly inefficient due to the existence of a blood–tumor barrier (BTB), which hampers tumor accumulation and uptake. Integrin α_vβ₃ is overexpressed on glioblastoma U87 cells and neovasculture, thus making its ligands such as the RGD motif target glioblastoma in vitro and in vivo. In the present work, we have designed a modified polyethylene glycol–polyethylenimine (PEG–PEI) gene carrier by conjugating it with a cyclic RGD sequence, c(RGDyK) (cyclic arginine‐glycine‐aspartic acid‐D ‐tyrosine‐lysine). When complexed with plasmid DNA, this gene carrier, termed RGD–PEG–PEI, formed homogenous nanoparticles with a mean diameter of 73 nm. These nanoparticles had a high binding affinity with U87 cells and facilitated targeted gene delivery against intracranial glioblastoma in vivo, thereby leading to a higher gene transfer efficiency compared to the PEG–PEI gene carrier without RGD decoration. This intracranial glioblastoma‐targeted gene carrier also enhanced the therapeutic efficacy of pORF‐hTRAIL, as evidenced by a significantly prolonged survival of intracranial glioblastoma‐bearing nude mice. Considering the contribution of glioblastoma neovasculature to the BBB under angiogenic conditions, our results demonstrated the therapeutic feasibility of treating a brain tumor through mediation of integrin α_vβ₃, as well as the potential of using RGD–PEG–PEI as a targeted gene carrier in the treatment of intracranial glioblastoma.     

5‐Aminolevulinic acid‐Loaded Fullerene Nanoparticles for In Vitro and In Vivo Photodynamic Therapy

This report explores some properties of 80–200 nm nanoparticles containing 5‐aminolevulinic acid (ALA) and fullerene (C60) for photodynamic therapy (PDT). Compared with ALA, the nanoparticles yielded more protoporphyrin IX (PpIX) formation in cells and tissues and to a significant improvement in antitumor efficacy in tumor‐bearing mice. Maximum levels of PpIX were obtained 4 h after administration and selective PpIX formation in tumor was observed. These nanoparticles appear to be a useful vehicle for drug delivery purposes. In this study, a procedure for preparing fullerene nanoparticles containing ALA was developed. The product alone exhibited no detectable toxicity in the dark and was superior to ALA alone in promoting PpIX biosynthesis and PDT efficacy both in culture and in a murine tumor model. These results suggest that this procedure could be the basis for an improved PDT protocol for cancer control.     

Development of a ZHER3‐Affibody‐Targeted Nano‐Vector for Gene Delivery to HER3‐Overexpressed Breast Cancer Cells

Despite the initial successes of gene delivery applications, they faced on several intrinsic drawbacks including toxicity and immunogenicity. Therefore, alternative gene‐delivery systems derived from recombinant peptides have emerged and is rapidly developing. Human epidermal growth factor receptor‐3 (HER3) shows high activity in tumor resistance to anti‐human epidermal growth factor receptor 2 (HER2) therapies. In this study, an affibody molecule against HER3 is conjugated to a biomimetic peptide RALA (an amphipathic and cationic peptide enriched with arginine) and the ability of the fusion vector for targeting HER3 and afterward delivering specific genes in breast cancer cells is evaluated. The results demonstrate that the biopolymeric platform, which contains an affibody‐conjugated RALA peptide, can effectively condense DNA into nanoparticles and target the overexpressed HER3 receptors in breast cancer cells and transfer specific genes. The use of such a recombinant biopolymer may pave the way for the development of sensitive and effective diagnostic and treatment tool for breast cancer.     

Polyethylenimine‐Based Nanogels for Biomedical Applications

Nanogels (NGs) are 3‐dimensional (3D) networks composed of hydrophilic or amphiphilic polymer chains, allowing for effective and homogeneous encapsulation of drugs, genes, or imaging agents for biomedical applications. Polyethylenimine (PEI), possessing abundant positively charged amine groups, is an ideal platform for the development of NGs. A variety of effective PEI‐based NGs have been designed and much effort has been devoted to study the relationship between the structure and function of the NGs. In particular, PEI‐based NGs can be prepared either using PEI as the major NG component or using PEI as a crosslinker. This review reports the recent progresses in the design of PEI‐based NGs for gene and drug delivery and for bioimaging applications with a target focus to tackle the diagnosis and therapy of cancer.     

Photodynamic Therapy with Tumor Cell Discrimination through RNA-Targeting Ability of Photosensitizer

Photodynamic therapy (PDT) represents an effective treatment to cure cancer. The targeting ability of the photosensitizer is of utmost importance. Photosensitizers that discriminate cancer cells can avoid the killing of normal cells and improve PDT efficacy. However, the design and synthesis of photosensitizers conjugated with a recognition unit of cancer cell markers is complex and may not effectively target cancer. Considering that the total RNA content in cancer cells is commonly higher than in normal cells, this study has developed the photosensitizer QICY with RNA-targeting abilities for the discrimination of cancer cells. QICY was specifically located in cancer cells rather than normal cells due to their stronger electrostatic interactions with RNA, thereby further improving the PDT effects on the cancer cells. After intravenous injection into mice bearing a xenograft tumor, QICY accumulated into the tumor location through the enhanced permeability and retention effect, automatically targeted cancer cells under the control of RNA, and inhibited tumor growth under 630 nm laser irradiation without obvious side effects. This intelligent photosensitizer with RNA-targeting ability not only simplifies the design and synthesis of cancer-cell-targeting photosensitizers but also paves the way for the further development of highly efficient PDTs.     

Programming DNA Nanoassembly for Enhanced Photodynamic Therapy

Photodynamic therapy (PDT) has extraordinary promise for the treatment of many cancers. However, its clinical progress is impaired by the intrinsic hypoxic tumor microenvironment that limits PDT efficacy and the safety concern associated with biological specificity of photosensitizers or vehicles. Now it is demonstrated that rationally designed DNA nanosponges can load and delivery photosensitizer effectively, target tumor precisely, and relieve hypoxia‐associated resistance remarkably to enhance the efficacy of PDT. Specifically, the approach exhibits a facile assembly process, provides programmable and versatile nanocarriers, and enables robust in vitro and in vivo anti‐cancer efficacy with excellent biosafety. These findings represent a practical and safe approach by designer DNA nanoassemblies to combat cancer effectively and suggest a powerful strategy for broad biomedical application of PDT.     

Improvement of Targeted Gene Delivery to Human Cancer Cells by a Novel Trifunctional Crosslinker

A facile method for the construction of an immunoconjugate which displays targeting ligands, such as antibody fragments, with a high density is reported. For this purpose, we synthesized a novel trifunctional crosslinking reagent. By the use of this reagent, ligands targeting the specific cell can be displayed on the surface of the drug carrier with a high density. In this study, we display HER2 (human epidermal growth‐factor receptor‐2) binding ligands on branched polyethylenimine (PEI), which can form polyplexes with plasmid DNA. Kinetic analysis of the binding to the extracellular domain of HER2 show the PEI displaying a high density of ligands binds to the target more strongly compared to the PEI displaying ligands at a low density. The increased density of HER2 ligands displayed on the gene carrier contributes to the improved transfection efficiency. This approach can be applied to other drug delivery systems, including liposome, micelle, and so on.     

Biodegradable Inorganic Nanovector: Passive versus Active Tumor Targeting in siRNA Transportation

The biodegradable inorganic nanovector based on a layered double hydroxide (LDH) holds great promise for gene and drug delivery systems. However, in vivo targeted delivery of genes through LDH still remains a key challenge in the development of RNA interference therapeutics. Here, we describe in vivo and in vitro delivery system for Survivin siRNA (siSurvivin) assembled with passive LDH with a particle size of 100 nm or active LDH conjugated with a cancer overexpressing receptor targeting ligand, folic acid (LDHFA), conferring them an ability to target the tumor by either EPR‐based clathrin‐mediated or folate receptor‐mediated endocytosis. When not only transfected into KB cells but also injected into xenograft mice, LDHFA/siSurvivin induced potent gene silencing at mRNA and protein levels in vitro, and consequently achieved a 3.0‐fold higher suppression of tumor volume than LDH/siSurvivin in vivo. This anti‐tumor effect was attributed to a selectively 1.2‐fold higher accumulation of siSurvivin in tumor tissue compared with other organs. Targeting to the tumor with inorganic nanovector can guide and accelerate an evolution of next‐generation theranosis system.     

(Coixan polysaccharide)-graft-polyethylenimine folate for tumor-targeted gene delivery

CP-PEI-FA was prepared as an effective vector for in vitro and in vivo tumor-targeted gene delivery. The structures of the polymers were characterized, and their DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro/in vivo transfection were examined. The cytotoxicity of CP-PEI-FA was significantly lower than that of PEI 25 kDa and close to that of PEI 1200. The in vitro transfection of CP-PEI-FA was tested in C6 and HeLa cells (FR-positive cells) and A549 cells (FR-negative cells). CP-PEI-FA showed a high targeting specificity and good gene transfection efficiency in FR-positive cells. These results indicate that CP-PEI-FA is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA.     

An Assembled Nanocomplex for Improving both Therapeutic Efficiency and Treatment Depth in Photodynamic Therapy

Photodynamic therapy (PDT) shows unique selectivity and irreversible destruction toward treated tissues or cells, but still has several problems in clinical practice. One is limited therapeutic efficiency, which is attributed to hypoxia in tumor sites. Another is the limited treatment depth because traditional photosensitizes are excited by short wavelength light (<700 nm). An assembled nano‐complex system composed of oxygen donor, two‐photon absorption (TPA) species, and photosensitizer (PS) was synthesized to address both problems. The photosensitizer is excited indirectly by two‐photon laser through intraparticle FRET mechanism for improving treatment depth. The oxygen donor, hemoglobin, can supply extra oxygen into tumor location through targeting effect for enhanced PDT efficiency. The mechanism and PDT effect were verified through both in vitro and in vivo experiments. The simple system is promising to promote two‐photon PDT for clinical applications.     

Photodynamic Therapy and the Biophysics of the Tumor Microenvironment

Targeting the tumor microenvironment (TME) provides opportunities to modulate tumor physiology, enhance the delivery of therapeutic agents, impact immune response and overcome resistance. Photodynamic therapy (PDT) is a photochemistry-based, nonthermal modality that produces reactive molecular species at the site of light activation and is in the clinic for nononcologic and oncologic applications. The unique mechanisms and exquisite spatiotemporal control inherent to PDT enable selective modulation or destruction of the TME and cancer cells. Mechanical stress plays an important role in tumor growth and survival, with increasing implications for therapy design and drug delivery, but remains understudied in the context of PDT and PDT-based combinations. This review describes pharmacoengineering and bioengineering approaches in PDT to target cellular and noncellular components of the TME, as well as molecular targets on tumor and tumor-associated cells. Particular emphasis is placed on the role of mechanical stress in the context of targeted PDT regimens, and combinations, for primary and metastatic tumors.     

MC8 Peptide-Mediated Her-2 Receptor Targeting Based on PEI-β-CyD as Gene Delivery Vector

A novel vector with high gene delivery efficiency and special cell targeting ability was developed using a good strategy that utilized low molecular weight polyethylenimine (PEI; molecular weight, 600 KDa [PEI600]) cross-linked to β-cyclodextrin (β-CyD) via a facile synthetic route. Human epidermal growth factor receptor 2 (Her-2) are highly expressed in a variety of human cancer cells and are potential targets for cancer therapy. MC8 peptides, which have been proven to combine especially with Her-2 on cell membranes were coupled to PEI-β-CyD using N-succinimidyl-3-(2-pyridyldithio) propionate as a linker. The ratios of PEI600, β-CyD, and peptide were calculated based on proton integral values obtained from the ¹H-NMR spectra of the resulting products. Electron microscope observations showed that MC8-PEI-β-CyD can efficiently condense plasmid DNA (pDNA) into nanoparticles of about 200 nm, and MTT assays suggested the decreased toxicity of the polymer. Experiments on gene delivery efficiency in vitro showed that MC8-PEI-β-CyD/pDNA polyplexes had significantly greater transgene activities than PEI-β-CyD/pDNA in the Skov3 and A549 cells, which positively expressed Her-2, whereas, no such effect was observed in the MCF-7 cells, which negatively expressed Her-2. Our current research indicated that the synthesized nonviral vector shows improved gene delivery efficiency and targeting specificity in Her-2 positive cells.