Photodynamically Tumor Vessel Destruction Amplified Tumor Targeting of Nanoparticles for Efficient Chemotherapy.

Efficient tumor-targeted drug delivery is still a challenging and currently unbreakable bottleneck in chemotherapy for tumors. Nanomedicines based on passive or active targeting strategy have not yet achieved convincing chemotherapeutic benefits in the clinic due to the tumor heterogeneity. Inspired by the efficient inflammatory-cell recruitment to acute clots, we constructed a two-component nanosystem, which is composed of an RGD-modified pyropheophorbide-a (Ppa) micelle (PPRM) that mediates the tumor vascular-targeted photodynamic reaction to activate local coagulation and subsequently transmits the coagulation signals to the circulating clot-targeted CREKA peptide-modified camptothecin (CPT)-loaded nanodiscs (CCNDs) for amplifying tumor targeting. PPRM could effectively bind with the tumor vasculature and induce sufficient local thrombus by a photodynamic reaction. Local photodynamic reaction-induced tumor target amplification greatly increased the tumor accumulation of CCND by 4.2 times, thus significantly enhancing the chemotherapeutic efficacy in the 4T1 breast tumor model. In other words, this study provides a powerful platform to amplify tumor-specific drug delivery by taking advantage of the efficient crosstalk between the PPRM-activated coagulation cascade and clot-targeted CCND.

Manipulating Redox Homeostasis of Cancer Stem Cells Overcome Chemotherapeutic Resistance through Photoactivatable Biomimetic Nanodiscs.

Tumor heterogeneity remains a significant obstacle in cancer therapy due to diverse cells with varying treatment responses. Cancer stem-like cells (CSCs) contribute significantly to intratumor heterogeneity, characterized by high tumorigenicity and chemoresistance. CSCs reside in the depth of the tumor, possessing low reactive oxygen species (ROS) levels and robust antioxidant defense systems to maintain self-renewal and stemness. A nanotherapeutic strategy is developed using tumor-penetrating peptide iRGD-modified high-density lipoprotein (HDL)-mimetic nanodiscs (IPCND) that ingeniously loaded with pyropheophorbide-a (Ppa), bis (2-hydroxyethyl) disulfide (S-S), and camptothecin (CPT) by synthesizing two amphiphilic drug-conjugated sphingomyelin derivatives. Photoactivatable Ppa can generate massive ROS which as intracellular signaling molecules effectively shut down self-renewal and trigger differentiation of the CSCs, while S-S is utilized to deplete GSH and sustainably imbalance redox homeostasis by reducing ROS clearance. Simultaneously, the depletion of GSH is accompanied by the release of CPT, which leads to subsequent cell death. This dual strategy successfully disturbed the redox equilibrium of CSCs, prompting their differentiation and boosting the ability of CPT to kill CSCs upon laser irradiation. Additionally, it demonstrated a synergistic anti-cancer effect by concurrently eliminating therapeutically resistant CSCs and bulk tumor cells, effectively suppressing tumor growth in CSC-enriched heterogeneous colon tumor mouse models.

Pyroptosis Induction with Nanosonosensitizer-Augmented Sonodynamic Therapy Combined with PD-L1 Blockade Boosts Efficacy against Liver Cancer.

Induction of pyroptosis can promote anti-PD-L1 therapeutic efficacy due to the release of pro-inflammatory cytokines, but current approaches can cause off target toxicity. Herein, a phthalocyanine-conjugated mesoporous silicate nanoparticle (PMSN) is designed for amplifying sonodynamic therapy (SDT) to augment oxidative stress and induce robust pyroptosis in tumors. The sub-10 nm diameter structure and c(RGDyC)-PEGylated modification enhance tumor targeting and renal clearance. The unique porous architecture of PMSN doubles ROS yield and enhances pyroptotic cell populations in tumors (25.0%) via a cavitation effect. PMSN-mediated SDT treatment efficiently reduces tumor mass and suppressed residual tumors in treated and distant sites by synergizing with PD-L1 blockade (85.93% and 77.09%, respectively). Furthermore, loading the chemotherapeutic, doxorubicin, into PMSN intensifies SDT-pyroptotic effects and increased efficacy. This is the first report of the use of SDT regimens to induce pyroptosis in liver cancer. This noninvasive and effective strategy has potential for clinical translation.

Manipulating Neovasculature-Targeting Capability of Biomimetic Nanodiscs for Synergistic Photoactivatable Tumor Infarction and Chemotherapy.

Tumor infarction therapy is a promising antitumor strategy with the advantages of taking a short therapy duration, less risk of resistance, and effectiveness against a wide range of tumor types. However, its clinical application is largely hindered by tumor recurrence in the surviving rim and the potential risk of thromboembolic events due to nonspecific vasculature targeting. Herein, a neovasculature-targeting synthetic high-density lipoprotein (sHDL) nanodisc loaded with pyropheophorbide-a and camptothecin (CPN) was fabricated for photoactivatable tumor infarction and synergistic chemotherapy. By manipulating the anisotropy in ligand modification of sHDL nanodiscs, CPN modified with neovaculature-targeting peptide on the planes (PCPN) shows up to 7-fold higher cellular uptake compared with that around the edge (ECPN). PCPN can efficiently bind to endothelial cells of tumor vessels, and upon laser irradiation, massive local thrombus can be induced by the photodynamic reaction to deprive nutrition supply. Meanwhile, CPT could be released in response to the tumor reductive environment, thus killing residual tumor cells in the surviving rim to inhibit recurrence. These findings not only offer a powerful approach of synergistic cancer therapy but also suggest the potential of plane-modified sHDL nanodiscs as a versatile drug delivery nanocarrier.

Major Strategies for Spatial Control of Ultrasound-Driven Gene Expression to Enhance Therapeutic Specificity.

A major challenge of gene therapy is to achieve highly specific transgene expression in tissues of interest with minimized off-target expression. Ultrasound in combination with microbubbles can transiently increase permeability of desired cells or tissues and thereby facilitate gene transfer. This kind of ultrasound-driven transgene expression has gained increasing attention due to its deep tissue penetration and high spatiotemporal resolution. However, successful genetic manipulation in vivo with ultrasound need to well optimize various aspects involved in this process. Ultrasound parameters, microbubble dose, and gene vectors need to be optimized for highly increased transgene expression in the cells of interest. Conversely, the potential off-target transgene expression and toxicities need to be reduced by modification of gene vectors and/or promoter sequence. This review will discuss some major strategies for enhanced specificity of the ultrasound-mediated gene transfer in vivo. Five major strategies will be discussed, including the integration of real-time imaging methods, local injection, targeted microbubbles loaded with nucleic acids, stealth nanocarriers, and cell-specific promoter. The advantages and limitations of each strategy were outlined, hoping to provide a guideline for researchers in achieving high specific ultrasound-driven gene expression.

Enhancing Photodynamic Therapy Efficacy Against Cancer Metastasis by Ultrasound-Mediated Oxygen Microbubble Destruction to Boost Tumor-Targeted Delivery of Oxygen and Renal-Clearable Photosensitizer Micelles.

Hypoxic tumor microenvironment and nonspecific accumulation of photosensitizers are two key factors that limit the efficacy of photodynamic therapy (PDT). Herein, a strategy of oxygen microbubbles (MBs) boosting photosensitizer micelles is developed to enhance PDT efficacy and inhibit tumor metastasis by self-assembling renal-clearable ultrasmall poly(ethylene glycol)-modified protoporphyrin IX micelles (PPM) and perfluoropentane (PFP)-doped oxygen microbubbles (OPMBs), followed by ultrasound imaging-guided OPMB destruction to realize the tumor-targeted delivery of PPM and oxygen in tumor. Doping PFP into oxygen MBs increases the production of MBs and stability of oxygen MBs, allowing for persistent circulation in blood. Following co-injection, destruction of OPMBs with ultrasound leads to ∼2.2-fold increase of tumor-specific PPM accumulation. Furthermore, the burst release of oxygen by MB destruction improves tumor oxygenation from 22 to 50%, which not only raises the production of singlet oxygen but also significantly reduces the expression of hypoxia-inducible factor-1 alpha and related genes, thus preventing angiogenesis and epithelial-mesenchymal transition. It is verified that this strategy effectively eradicates orthotopic breast cancer and inhibits lung metastasis. Furthermore, the survival rate of mice bearing orthotopic pancreatic tumor is significantly extended by such interventional PDT strategy. Therefore, the combination of ultrasmall PPM and OPMBs represents a simple but effective strategy in overcoming the limitations of PDT.

Photosensitizer Nanoparticles Boost Photodynamic Therapy for Pancreatic Cancer Treatment.

Patients with pancreatic cancer (PCa) have a poor prognosis apart from the few suitable for surgery. Photodynamic therapy (PDT) is a minimally invasive treatment modality whose efficacy and safety in treating unresectable localized PCa have been corroborated in clinic. Yet, it suffers from certain limitations during clinical exploitation, including insufficient photosensitizers (PSs) delivery, tumor-oxygenation dependency, and treatment escape of aggressive tumors. To overcome these obstacles, an increasing number of researchers are currently on a quest to develop photosensitizer nanoparticles (NPs) by the use of a variety of nanocarrier systems to improve cellular uptake and biodistribution of photosensitizers. Encapsulation of PSs with NPs endows them significantly higher accumulation within PCa tumors due to the increased solubility and stability in blood circulation. A number of approaches have been explored to produce NPs co-delivering multi-agents affording PDT-based synergistic therapies for improved response rates and durability of response after treatment. This review provides an overview of available data regarding the design, methodology, and oncological outcome of the innovative NPs-based PDT of PCa.

Key considerations in designing CRISPR/Cas9-carrying nanoparticles for therapeutic genome editing.

CRISPR-Cas9, the breakthrough genome-editing technology, has emerged as a promising tool to prevent and cure various diseases. The efficient genome editing technology strongly relies on the specific and effective delivery of CRISPR/Cas9 cargos. However, the lack of a safe, specific, and efficient non-viral delivery system for in vivo genome editing remains a major limit for its clinical translation. In this review, we will first briefly introduce the working mechanism of CRISPR/Cas9 and the patterns of CRISPR/Cas9 delivery. Furthermore, the physiological obstacles for the delivery process in vivo are elaborated. Finally, the key considerations will be deeply discussed in designing non-viral nanovectors for therapeutic CRISPR/Cas9 delivery in vivo, including the effective encapsulation of large-size macromolecules, targeting specific tissues and cells, efficient endosomal escape and safety concerns of the vector systems, in the hope of inviting more comprehensive studies on the development of safe, specific, and efficient non-viral nanovectors for delivering a CRISPR/Cas9 system.

Bioluminescence Imaging of Inflammation in Vivo Based on Bioluminescence and Fluorescence Resonance Energy Transfer Using Nanobubble Ultrasound Contrast Agent.

Inflammation is an immunological response involved in various inflammatory disorders ranging from neurodegenerative diseases to cancers. Luminol has been reported to detect myeloperoxidase (MPO) activity in an inflamed area through a light-emitting reaction. However, this method is limited by low tissue penetration and poor spatial resolution. Here, we fabricated a nanobubble (NB) doped with two tandem lipophilic dyes, red-shifting luminol-emitted blue light to near-infrared region through a process integrating bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET). This BRET-FRET process caused a 24-fold increase in detectable luminescence emission over luminol alone in an inflammation model induced by lipopolysaccharide. In addition, the echogenicity of the BRET-FRET NBs also enables perfused tissue microvasculature to be delineated by contrast-enhanced ultrasound imaging with high spatial resolution. Compared with commercially available ultrasound contrast agent, the BRET-FRET NBs exhibited comparable contrast-enhancing capability but much smaller size and higher concentration. This bioluminescence/ultrasound dual-modal contrast agent was then successfully applied for imaging of an animal model of breast cancer. Furthermore, biosafety experiments revealed that multi-injection of luminol and NBs did not induce any observable abnormality. By integrating the advantages of bioluminescence imaging and ultrasound imaging, this BRET-FRET system may have the potential to address a critical need of inflammation imaging.

Intraoperative Identification and Guidance of Breast Cancer Microfoci Using Ultrasound and Near-Infrared Fluorescence Dual-Modality Imaging.

The traditional method of labeling the nonpalpable breast cancer is placing a guidewire or metal marker guided by ultrasound or stereographic mammogram prior to surgery. However, the wire localization has a risk of displacement and could be an obstacle in the surgical course. To avoid these issues, we tried to combine the near-infrared (NIR) fluorescence dye dioctadecyltetramethyl indotricarbocyanine iodide (DiR) and microbubbles (MBs) to realize the dual-modality imaging for breast cancer microfoci intraoperative identification and guidance as a more efficient workflow. First, 24 mice were divided into three groups, injected with DiR nanoparticles (NPs), DiR MBs, and DiR MBs + ultrasound (US), and then, in vivo and ex vivo NIR fluorescence imaging was conducted. The distinction of fluorescence imaging intensity at the tumor site among the three groups was statistically significant (P < 0.001). Group 3 (DiR MBs + US) exhibited the highest fluorescence imaging intensity; the distinctions between group 3 and group 1 (DiR NPs) and group 3 and group 2 (DiR MBs) were both statistically significant (P = 0.001, P = 0.003), while the distinction between group 1 and group 2 was not statistically significant (P = 1.0). The results above validated the advantage of fluorescence imaging by using ultrasound-targeted MB destruction. Second, two kinds of subcutaneous breast cancer mice models [4T1-luc(n = 5)/MCF-7(n = 3)] received tumor resection, and NIR fluorescence and bioluminescence images were obtained to detect tumor residuals. We found that the small residual tumor tissues, metastatic lymph nodes, and even the surrounding infiltrated tissue all can be indicated by the fluorescence imaging and verified with bioluminescence and histological examination. In addition, the residual tumor cells appeared as tumor recurrence 22 days post operation and was confirmed with contrast-enhanced ultrasound (CEUS) in vivo. Thereby, ultrasound-targeted DiR MB destruction and then conversion into DiR NPs was feasible for intraoperative identification and guidance of nonpalpable breast cancer foci.

Self-assembly of porphyrin-grafted lipid into nanoparticles encapsulating doxorubicin for synergistic chemo-photodynamic therapy and fluorescence imaging.

The limited clinical efficacy of monotherapies in the clinic has urged the development of novel combination platforms. Taking advantage of light-triggered photodynamic treatment combined together with the controlled release of nanomedicine, it has been possible to treat cancer without eliciting any adverse effects. However, the challenges imposed by limited drug loading capacity and complex synthesis process of organic nanoparticles (NPs) have seriously impeded advances in chemo-photodynamic combination therapy. In this experiment, we utilize our previously synthesized porphyrin-grafted lipid (PGL) NPs to load highly effective chemotherapeutic drug, doxorubicin (DOX) for synergistic chemo-photodynamic therapy. Methods: A relatively simple and inexpensive rapid injection method was used to prepare porphyrin-grafted lipid (PGL) NPs. The self-assembled PGL NPs were used further to encapsulate DOX via a pH-gradient loading protocol. The self-assembled liposome-like PGL NPs having a hydrophilic core were optimized to load DOX at an encapsulation efficiency (EE) of ~99%. The resultant PGL-DOX NPs were intact, highly stable and importantly these NPs successfully escaped from the endo-lysosomal compartment after laser irradiation to release DOX in the cytosol. The therapeutic efficacy of the aforementioned formulation was validated both in vitro and in vivo. Results: PGL-DOX NPs demonstrated excellent cellular uptake, chemo-photodynamic response, and fluorescence imaging ability in different cell lines. Under laser irradiation, cells treated with a low molar concentration of PGL-DOX NPs reduced cell viability significantly. Moreover, in vivo experiments conducted in a xenograft mouse model further demonstrated the excellent tumor accumulation capability of PGL-DOX NPs driven by the enhanced permeability and retention (EPR) effect. Through fluorescence imaging, the biodistribution of PGL-DOX NPs in tumor and major organs was also easily monitored in real time in vivo. The inherent ability of porphyrin to generate ROS under laser irradiation combined with the cytotoxic effect of the anticancer drug DOX significantly suppressed tumor growth in vivo. Conclusion: In summary, the PGL-DOX NPs combined chemo-photodynamic nanoplatform may serve as a potential candidate for cancer theranostics.

Ultrasound-targeted photodynamic and gene dual therapy for effectively inhibiting triple negative breast cancer by cationic porphyrin lipid microbubbles loaded with HIF1α-siRNA.

Triple-negative breast cancer (TNBC), which is a kind of aggressive breast cancer, has a much higher recurrence rate and a shorter overall survival rate than other breast cancer subtypes owing to its lack of expression of the progesterone receptor (PR), estrogen receptor (ER) and HER2. For improving the therapeutic efficacy of TNBC, we developed a new kind of multifunctional cationic porphyrin-grafted lipid (CPGL) microbubble loaded with HIF 1α siRNA (siHIF@CpMB). Owing to the amphiphilic structure of CPGL, it can be self-assembled into microbubbles (MBs) with conventional lipids and the porphyrin group could be used as a photosensitizer for photodynamic therapy (PDT), while the amino group could adsorb HIF 1α siRNA (siHIF) through electrostatic adsorption. Such MBs possess a remarkably high drug loading content and less premature drug release. Distribution of MBs could be easily monitored by real-time US imaging (3-12 MHz). Furthermore, with the assistance of ultrasound targeted microbubble destruction (UTMD), siHIF@CpMBs could be efficiently converted into nanoparticles in situ, facilitating the accumulation of porphyrin and siRNA at the tumor site through the cavitation effect. HIF 1α siRNA down-regulated the HIF 1α level, which was induced by the common hypoxic tumor environment or the ROS (generated by PDT), enhanced the PDT efficacy and partly inhibited the tumor progression. Therefore, UTMD assisted combination of PDT and gene therapy was believed to be an effective therapeutic strategy for TNBC.

Localized delivery of curcumin into brain with polysorbate 80-modified cerasomes by ultrasound-targeted microbubble destruction for improved Parkinson's disease therapy.

Rationale: Treatment for Parkinson's disease (PD) is challenged by the presence of the blood-brain barrier (BBB) that significantly limits the effective drug concentration in a patient's brain for therapeutic response throughout various stages of PD. Curcumin holds the potential for α-synuclein clearance to treat PD; however, its applications are still limited due to its low bioavailability and poor permeability through the BBB in a free form. Methods: Herein, this paper fabricated curcumin-loaded polysorbate 80-modified cerasome (CPC) nanoparticles (NPs) with a mean diameter of ~110 nm for enhancing the localized curcumin delivery into the targeted brain nuclei via effective BBB opening in combination with ultrasound-targeted microbubble destruction (UTMD). Results: The liposomal nanohybrid cerasome exhibited superior stability towards PS 80 surfactant solubilization and longer circulation lifetime (t1/2 = 6.22 h), much longer than free curcumin (t1/2 = 0.76 h). The permeation was found to be 1.7-fold higher than that of CPC treatment only at 6 h after the systemic administration of CPC NPs. Notably, motor behaviors, dopamine (DA) level and tyrosine hydroxylase (TH) expression all returned to normal, thanks to α-synuclein (AS) removal mediated by efficient curcumin delivery to the striatum. Most importantly, the animal experiment demonstrated that the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice had notably improved behavior disorder and dopamine depletion during two-week post-observation after treatment with CPC NPs (15 mg curcumin/kg) coupled with UTMD. Conclusion: This novel CPC-UTMD formulation approach could be an effective, safe and amenable choice with higher therapeutic relevance and fewer unwanted complications than conventional chemotherapeutics delivery systems for PD treatment in the near future.

Nano-sized Indocyanine Green J-aggregate as a One-component Theranostic Agent.

The development of biocompatiable efficient photothermal coupling agent (PCA) for image-guided photothermal therapy of cancer has gained increasing interests in recent years. Although various PCAs have been developed, the clinical translations of these materials have been largely hindered by the potential biosafety issues and challenges of scaling-up manufactures. In this research, we proposed nano-sized indocyanine green (ICG) J-aggregate (IJA) as a promising PCA which is fabricated by a very facile method using clinical-approved ICG as the only excipient. The as-prepared IJA remains stable in various solution and shows a ~115 nm red-shift in absorption peak compared to free ICG. Importantly, IJA can be disassociated into free ICG again after internalized into cells and exhibits high biosafety comparable to ICG. The IJA performs well for photothermal therapy both in vitro and in vivo. In addition, the IJA can also be used as a good photoacoustic contrast agent and internalization-responsive fluorescence probe. The facile preparation, high safety and excellent theranostic performance indicated that IJA might be a promising one-component agent for cancer theranostics.

Doxorubicin and Indocyanine Green Loaded Hybrid Bicelles for Fluorescence Imaging Guided Synergetic Chemo/Photothermal Therapy.

Hybrid bicelles have been demonstrated to have great potential for hydrophobic drug delivery. Herein, we report a near-infrared light-driven, temperature-sensitive hybrid bicelles co-encapsulating hydrophobic doxorubicin (DOX) and indocyanine green (ICG) (DOX/ICG@HBs). Encapsulation of ICG into the lipid bilayer membrane of DOX/ICG@HBs results in higher photostability than free ICG. DOX/ICG@HBs exhibited temperature-regulated drug release behavior and significant photothermal cytotoxicity. After tail vein injection, such discotic nanoparticles of DOX/ICG@HBs were found to accumulate selectively at the tumor site and act as an efficient probe to enhance fluorescence imaging greatly. The in vivo experiments showed that the DOX/ICG@HBs-mediated chemo- and photothermal combination therapy was more cytotoxic to tumor cells than the photothermal treatment or the chemotherapy alone due to the synergistic effect, reducing the occurrence of tumor metastasis. Therefore, DOX/ICG@HBs can act as a powerful nanotheranostic agent for chemo/photothermal therapy of cancer under the guidance of near-infrared fluorescence imaging.

A Novel Cyanine and Porphyrin Based Theranostic Nanoagent for Near-Infrared Fluorescence Imaging Guided Synergistic Phototherapy.

Design and synthesis of multifunctional organic nanoparticles (NPs) for non-invasive diagnosis and phototherapy of cancer are widely acknowledged. However, limited drug loading of NPs are major limitations for attaining synergistic effect in most of the combination therapies. Herein, a novel cyanine-porphyrin combined NPs (PGL-DiR) was fabricated in a simple and inexpensive way for the treatment of prostate cancer. A most commonly used near-infrared fluorescent (NIRF) cyanine dye, DiR was chosen as a photothermal agent to encapsulate in a porphyrin grafted lipid (PGL) (PGL-DiR) NPs of average size 156.25±31.31 nm. Unlike conventional liposomes, the self-assembled PGL morphology revealed encapsulation efficiency of DiR higher than 98%. However, as observed in vitro, DiR loading contents could trigger obvious quenching of singlet oxygen (1O2) by PGL thereby reducing the efficiency of PDT. Nevertheless, after successful photobleaching of DiR, as prepared PGL-DiR NP could facilitate enhanced synergistic photothermal and photodynamic (PTT-PDT) therapy both in vitro and in vivo. After intravenous administration of PGL-DiR NPs in a PC3 tumor xenograft mice, a high accumulation in tumor until 24 hrs was clearly evident via NIRF imaging. With the successive laser irradiation, these NPs effectively suppressed the tumor growth synergistically as PTT < PDT < PTT + PDT while compared to monotherapies such as PTT or PDT alone. These results demonstrated that as prepared PGL-NPs could serve as an excellent theranostic agent both in vitro and in vivo for combined therapy of prostate cancer.