Spheroid Penetration Research Articles

Tumor micro-environment targeted collagenase-modified albumin nanoparticles for improved drug delivery

Gemcitabine is a deoxycytidine analogue used as an adjunct therapy in the treatment of various solid tumors. The therapeutic efficacy of this drug is compromised because of its short circulation half-life (∼17 min), fast metabolic degradation and dose dependent adverse effects. Also, there are various barriers in the tumor microenvironment which limit the drug delivery to the solid tumor. Keeping these aspects into consideration, in the present investigation, protease (collagenase) coated gemcitabine-loaded human serum albumin (HSA) nanoparticles (CG-HSANPs) were prepared to improve the drug delivery to the solid tumor compared with uncoated gemcitabine-loaded HSA nanoparticles (G-HSANPs). The desolvation technique was used to develop nanocarriers, which were evaluated for physicochemical properties such as average particle diameter, surface charge, morphology, drug loading and in vitro drug release. The average particle diameter of G-HSANPs and CG-HSANPs was found to be 160.5 ± 4.1 and 181.7 ± 2.54 nm, respectively. The morphology of nanocarrier was investigated by transmission electron microscopy (TEM) and the nanoparticles were also evaluated using differential scanning calorimetry (DSC). The in vitro antiproliferative activity showed comparable cytotoxicity of CG-HSANPs, G-HSANPs and native drug. The fluorescence microscopy showed uptake of collagenase coated HSA nanoparticles (C-HSANPs) and uncoated HSA nanoparticles (HSANPs) in MiaPaCa-2 cells and there was higher nuclear fragmentation, generation of reactive oxygen species and perturbation in mitochondrial membrane potential of CG-HSANPs compared to the native drug. Further, C-HSANPs exhibited improved tumor spheroid penetration as compare to HSANPs. The present study demonstrated the potential of CG-HSANPs as a carrier for improved drug delivery.

Cell Membrane-Camouflaged Nanocarriers with Biomimetic Deformability of Erythrocytes for Ultralong Circulation and Enhanced Cancer Therapy.

Despite considerable advancements in cell membrane-camouflaged nanocarriers to leverage natural cell functions, artificial nanocarriers that can accurately mimic both the biological and physical properties of cells are urgently needed. Herein, inspired by the important effect of the stiffness and deformability of natural red blood cells (RBCs) on their life span and flowing through narrow vessels, we report the construction of RBC membrane-camouflaged nanocarriers that can mimic RBCs at different life stages and study how the deformability of RBC-derived nanocarriers affects their biological behaviors. RBC membrane-coated elastic poly(ethylene glycol) diacrylate hydrogel nanoparticles (RBC-ENPs) simulating dynamic RBCs exhibited high immunocompatibility with minimum immunoglobulin adsorption in the surface protein corona, resulting in reduced opsonization in macrophages and ultralong circulation. Furthermore, RBC-ENPs can deform like RBCs and achieve excellent diffusion in tumor extracellular matrix, leading to improved multicellular spheroid penetration and tumor tissue accumulation. In mouse cancer models, doxorubicin-loaded RBC-ENPs demonstrated superior antitumor efficacy to the first-line chemotherapeutic drug PEGylated doxorubicin liposomes. Our work highlights that tuning the physical properties of cell membrane-derived nanocarriers may offer an alternative approach for the bionic design of nanomedicines in the future.

Folic acid-modified curcumin-loaded liposomes for breast cancer therapy

Breast cancer is the commonest cancer among women, with more than 2 million cases per year and a lethality rate of 17.7%. The treatment of breast cancer still being a challenge due to the high heterogeneity of tumor cells. In this scenario, the development of targeted nanosystems for drug delivery has been investigated. Thus, the aim of this study was the development and characterization of folic acid-modified curcumin-loaded liposomes (LIP-CCM-FA) for the treatment of breast cancer. For this purpose, LIP-CCM-FA and unmodified liposomes (LIP-CCM) were developed and characterized in vitro using MCF-7 cells culture in two-dimensional (2D) and three-dimensional (3D) models. LIP-CCM and LIP-CCM-FA showed low particles size (~ 138 nm), narrow polydispersity indexes (~ 0.140), negative zeta potential (~ −13 mV), and high drug encapsulation efficiency (> 73%). Furthermore, LIP-CCM-FA showed a significantly greater cytotoxicity effect when compared with free curcumin and LIP-CCM using 2D and 3D cell culture models. In addition, in vitro cellular uptake assays using 2D and 3D models indicated higher cellular internalization and spheroid penetration of LIP-CCM-FA when compared with free curcumin and LIP-CCM, suggesting the potential of folic acid modification for improving drug internalization in breast cancer cells through its recognition by folate receptors overexpressed. In summary, this novel nanosystem demonstrated a greater in vitro antitumoral effect due to its ability to permeate tissues and be specifically recognized by folate receptors.

Optimizing the Design of Blood-Brain Barrier-Penetrating Polymer-Lipid-Hybrid Nanoparticles for Delivering Anticancer Drugs to Glioblastoma.

Chemotherapy for glioblastoma multiforme (GBM) remains ineffective due to insufficient penetration of therapeutic agents across the blood-brain barrier (BBB) and into the GBM tumor. Herein, is described, the optimization of the lipid composition and fabrication conditions for a BBB- and tumor penetrating terpolymer-lipid-hybridnanoparticle (TPLN) for delivering doxorubicin (DOX) to GBM. The composition of TPLNs was first screened using different lipids based on nanoparticle properties and in vitro cytotoxicity by using 23 full factorial experimental design. The leading DOX loaded TPLNs (DOX-TPLN) were prepared by further optimization of conditions and used to study cellular uptake mechanisms, in vitro cytotoxicity, three-dimensional (3D) glioma spheroid penetration, and in vivo biodistribution in a murine orthotopic GBM model. Among various lipids studied, ethyl arachidate (EA) was found to provide excellent nanoparticle properties e.g., size, polydispersity index (PDI), zeta potential, encapsulation efficiency, drug loading, and colloidal stability, and highest anticancer efficacy for DOX-TPLN. Further optimized EA-based TPLNs were prepared with an optimal particle size (103.8 ± 33.4nm) and PDI (0.208 ± 0.02). The resultant DOX-TPLNs showed ~ sevenfold higher efficacy than free DOX against human GBM U87-MG-RED-FLuc cells in vitro. The interaction between the TPLNs and the low-density lipoprotein receptors also facilitated cellular uptake, deep penetration into 3D glioma spheroids, and accumulation into the in vivo brain tumor regions of DOX-TPLNs. This work demonstrated that the TPLN system can be optimized by rational selection of lipid type, lipid content, and preparation conditions to obtain DOX-TPLN with enhanced anticancer efficacy and GBM penetration and accumulation.

Post-functionalization of drug-loaded nanoparticles prepared by polymerization-induced self-assembly (PISA) with mitochondria targeting ligands.

Herein, the postfunctionalization of different non-fouling PISA particles, prepared from either poly(oligo ethylene glycol methyl ether methacrylate) (pPEGMA) and the anticancer drug PENAO (4-(N-(S-penicillaminylacetyl)amino)phenylarsenonous acid) or zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) and PENAO were reported. Both PISA particles were reacted with triphenylphosphonium (TPP) as mitochondria targeting units in order to evaluate the changes in cellular uptake or the toxicity of the conjugated arsenic drug. Attachment of TPP onto the PISA particles however was found not to enhance the mitochondrial accumulation, but it did influence overall the biological activity of pMPC-based particles in 2D and 3D cultured sarcoma SW982 cells. When TPP was conjugated to the pMPC PISA particles more cellular uptake as well as better spheroid penetration were observed, while TPP on PEG-based PISA had only little effect. It was hypothesized that TPP on the micelle surface may not be accessible enough to allow mitochondria targeting, but more structural investigations are required to elucidate this.

Hyaluronic Acid-Functionalized Nanomicelles Enhance SAHA Efficacy in 3D Endometrial Cancer Models.

Simple SummaryOne of the major limitations to cancer therapies are the side effects caused by the drug interacting with any tissue in the body. There is often a balance between patient health and effectively treating the disease. To by-pass this balancing act nanoparticles are being used to deliver therapeutics straight to the tumors, acting as “Trojan Horses”. Endometrial cancers are known to have more of the cell surface protein CD44 than healthy tissues. Here, to efficiently target endometrial cancer, hyaluronic acid, which naturally binds to the CD44 protein was attached to the surface of nanoparticles and tested on microtissues or spheroids to better model a tumor and understand drug delivery performance. We show that our hyaluronic acid-nanoparticle formulations improve drug effects and interact with the cancer cells more than without this targeting agent.Histone Deacetylase (HDAC) enzymes are upregulated in cancer leading to the development of HDAC inhibiting compounds, several of which are currently in clinical trials. Side effects associated with toxicity and non-specific targeting indicate the need for efficient drug delivery approaches and tumor specific targeting to enhance HDAC efficacy in solid tumor cancers. SAHA encapsulation within F127 micelles functionalized with a surface hyaluronic acid moiety, was developed to target endometrial cancer cells expressing elevated levels of CD44. In vitro viability and morphology analyses was conducted in both 2D and 3D models to assess the translational potential of this approach. Encapsulation enhanced SAHA delivery and activity, demonstrating increased cytotoxic efficacy in 2D and 3D endometrial cancer models. High-content imaging showed improved nanoparticle internalization in 2D and CD44 enhanced penetration in 3D models. In addition, the nano-delivery system enhanced spheroid penetration resulting in cell growth suppression, p21 associated cell cycle arrest, as well as overcoming the formation of an EMT associated phenotype observed in free drug treated type II endometrial cancer cells. This study demonstrates that targeted nanoparticle delivery of SAHA could provide the basis for improving its efficacy in endometrial cancer. Using 3D models for endometrial cancer allows the elucidation of nanoparticle performance and CD44 targeting, likely through penetration and retention within the tumor model.

A Deep Eutectic Solvent-Based Approach to Intravenous Formulation.

Clinically viable formulations of hydrophobic drugs, for example, chemotherapeutics, require strategies to promote sufficient drug solubilization. However, such strategies often involve the use of organic solvents that pose a significant risk in generating toxic, unstable products. Using verteporfin as a drug, a deep eutectic solvent (DES)-based approach to solvate drugs in a simple one-step process is reported. Lipoidal DES composed of choline and oleate is used to successfully solvate verteporfin, resulting in stable sub-100nm nanocomplexes. The nanocomplexes successfully demonstrate efficient cellular uptake as well as retention, tumor spheroid penetration, and tumor accumulation in vivo. Systemic administration of the formulation significantly inhibits the primary tumor growth and its lung metastasis in the orthotopic 4T1 murine breast tumor model. Collectively, biocompatible DES shows great potential as a novel material for intravenous formulation of chemotherapeutics.

Metal Phenolic Network-Integrated Multistage Nanosystem for Enhanced Drug Delivery to Solid Tumors.

Metal-phenolic networks (MPNs) are an emerging class of supramolecular surface modifiers with potential use in various fields including drug delivery. Here, the development of a unique MPN-integrated core-satellite nanosystem (CS-NS) is reported. The "core" component of CS-NS comprises a liposome loaded with EDTA (a metal ion chelator) in the aqueous core and DiR (a near-infrared photothermal transducer) in the bilayer. The "satellite" component comprises mesoporous silica nanoparticles (MSNs) encapsulating doxorubicin and is coated with a Cu2+ -tannic acid MPN. Liposomes and MSNs self-assemble into the CS-NS through adhesion mediated by the MPN. When irradiated with an 808nm laser, CS-NS liberated the entrapped EDTA, leading to Cu2+ chelation and subsequent disassembly of the core-satellite nanostructure. Photo-conversion from the large assembly to the small constituent particles proceeded within 5 min. Light-triggered CS-NS disassembly enhanced the carrier and cargo penetration and accumulation in tumor spheroids in vitro and in orthotopic murine mammary tumors in vivo. CS-NS is long circulating in the blood and conferred improved survival outcomes to tumor-bearing mice treated with light, compared to controls. These results demonstrate an MPN-integrated multistage nanosystem for improved solid tumor treatment.

Two-dimensional LDH nanodisks modified with hyaluronidase enable enhanced tumor penetration and augmented chemotherapy

The condensed tumor extracellular matrix (ECM) consisting of cross-linked hyaluronic acid (HA) is one of the key factors that result in the aberrant tumor microenvironment and severely impair drug delivery and tumor penetration. Herein, we report a simple design of a hyaluronidase (HAase)-modified layered double hydroxide (LDH) nanoplatform loaded with anticancer drug doxorubicin (DOX) for enhanced tumor penetration and augmented chemotherapy. In our approach, LDH nanodisks were synthesized via a co-precipitation method, modified with HAase by electrostatic attraction, and finally physically loaded with DOX. The formulated DOX/LDH-HAase complexes show a high DOX loading percentage of 34.2% with good colloidal stability, retain 86.1% of the enzyme activity, and release DOX in a pH-responsive manner having a faster release rate under slightly acidic tumor microenvironment than that under a physiological condition. With the catalytic activity of HAase to digest the HA nearby the cancer cells, the developed DOX/LDH-HAase complexes enable more significant uptake by cancer cells and penetration in 3-dimensional tumor spheroids than enzyme-free DOX/LDH complexes, thus displaying much better antitumor efficacy in vitro than the latter. The more significant tumor penetration and inhibition of the DOX/LDH-HAase complexes than that of the DOX/LDH complexes was further demonstrated by in vivo tumor imaging and therapeutic activity assessments. Our study suggests a unique nanomedicine platform combined with both anticancer drug and enzyme for improved tumor penetration and chemotherapy, which is promising for effective chemotherapy of different types of stroma-rich tumors.

Benchtop X-ray fluorescence imaging as a tool to study gold nanoparticle penetration in 3D cancer spheroids.

The use of nanomaterials to improve medical diagnostics and therapeutics has been rapidly increasing. Among these materials are gold nanoparticles, which can be functionalized to target specific cells, acting as nanovectors for drug delivery, enhanced contrast agents as well as other targeted therapies. Au nanoparticles are very useful as they selectively accumulate in tumour sites due to the enhanced permeability-retention effect. There is however little information about the spatial distribution of the nanoparticles within tumours, which might hinder efficient therapies. In this study, X-ray fluorescence was used to investigate the diffusion of gold nanoparticles in cancer cell spheroids mimicking true tumour growth. Functionalization of the nanoparticles has the effect of allowing better diffusion into and out of the spheroid, while those nanoparticles that are only partially covered rapidly formed aggregates. This clustering led to size exclusion during transport within the tumour, changing its distribution profile while greatly increasing the nanoparticle concentration.

Nitric Oxide-Driven Nanomotor for Deep Tissue Penetration and Multidrug Resistance Reversal in Cancer Therapy.

Poor permeation of therapeutic agents and multidrug resistance (MDR) in solid tumors are the two major challenges that lead to the failure of the current chemotherapy methods. Herein, a zero‐waste doxorubicin‐loaded heparin/folic acid/l‐arginine (HFLA‐DOX) nanomotor with motion ability and sustained release of nitric oxide (NO) to achieve deep drug penetration and effective reversal of MDR in cancer chemotherapy is designed. The targeted recognition, penetration of blood vessels, intercellular penetration, special intracellular distribution (escaping from lysosomes and accumulating in Golgi and nucleus), 3D multicellular tumor spheroids (3D MTSs) penetration, degradation of tumor extracellular matrix (ECM), and reversal of MDR based on the synergistic effects of the motion ability and sustained NO release performance of the NO‐driven nanomotors are investigated in detail. Correspondingly, a new chemotherapy mode called recognition‐penetration‐reversal‐elimination is proposed, whose effectiveness is verified by in vitro cellular experiments and in vivo animal tumor model, which can not only provide effective solutions to these challenges encountered in cancer chemotherapy, but also apply to other therapy methods for the special deep‐tissue penetration ability of a therapeutic agent.

NIR Imaging of the Integrin-Rich Head and Neck Squamous Cell Carcinoma Using Ternary Copper Indium Selenide/Zinc Sulfide-Based Quantum Dots.

Simple SummaryDespite improved patient outcomes in a range of cancers, the prognostic of head and neck cancers remain poor, with more than 507,000 deaths annually. The management of these cancers requires multidisciplinary treatments, including radiotherapy, chemotherapy, or targeted molecular therapy; however, surgical resection is one of the first-line treatments for patients with head and neck cancers. In order to increase the positive surgical margin rate, we proposed a tumor-specific near-infrared nanoprobe for fluorescence-guided surgery. We confirmed the high specificity of the proposed nanoprobe towards head and neck cancer cells using advanced 3D stroma-rich spheroids.The efficient intraoperative identification of cancers requires the development of the bright, minimally-toxic, tumor-specific near-infrared (NIR) probes as contrast agents. Luminescent semiconductor quantum dots (QDs) offer several unique advantages for in vivo cellular imaging by providing bright and photostable fluorescent probes. Here, we present the synthesis of ZnCuInSe/ZnS core/shell QDs emitting in NIR (~750 nm) conjugated to NAVPNLRGDLQVLAQKVART (A20FMDV2) peptide for targeting αvβ6 integrin-rich head and neck squamous cell carcinoma (HNSCC). Integrin αvβ6 is usually not detectable in nonpathological tissues, but is highly upregulated in HNSCC. QD-A20 showed αvβ6 integrin-specific binding in two-dimension (2D) monolayer and three-dimension (3D) spheroid in vitro HNSCC models. QD-A20 exhibit limited penetration (ca. 50 µm) in stroma-rich 3D spheroids. Finally, we demonstrated the potential of these QDs by time-gated fluorescence imaging of stroma-rich 3D spheroids placed onto mm-thick tissue slices to mimic imaging conditions in tissues. Overall, QD-A20 could be considered as highly promising nanoprobes for NIR bioimaging and imaging-guided surgery.

Ultrasound and microbubbles (USMB) potentiated doxorubicin penetration and distribution in 3D breast tumour spheroids

Ultrasound and microbubbles (USMB) has been shown to improve chemotherapy in vitro cell suspension and monolayer models. These models are limited in their ability to study the potential of USMB in enhancing drug penetration within a three-dimensional (3D) tissue model which can in part reproduce the organization of a microtumour recapitulating cell-cell interactions and diffusion of molecules through the tissue. The 3D tumour spheroid models are considered as bridge for filling the gap between 2D in vitro monolayer cultured cells and animal models. In this study, we developed a 3D MDA-MB-231 breast cancer spheroid model and investigated the potential of USMB in enhancing drug penetration and distribution within the spheroid. The 3D spheroids were exposed to USMB (770 kPa Peak negative pressure, 1 MHz pulse frequency, 8 μs pulse duration and 2 m s pulse repetition period; and Definity at 1.7% v/v concentration) and doxorubicin (Dox) at 50 μM concentration. The short- and long-term effects on the spheroids were assessed using flow cytometry and Laser scanning confocal microscopy (LSCM). The application of USMB enhanced penetration of Dox into deeper regions of tumour spheroid. The Dox distribution was assessed using 3D optical sectioning and radial profiling at different z-planes of the spheroids. The flow cytometry data confirms that the USMB increases the Dox uptake per cell by 25% with 1.2 fold increase in total Dox fluorescence. No significant differences were observed in the long-term spheroid size with Dox + USMB compared to Dox alone. However, the spheroids appear more dispersed with the combined treatment of Dox + USMB compared to Dox alone. The tumour model show the enhanced chemotherapeutic effect in 3D tumour spheroids of USMB along with increased penetration and accumulation of chemotherapeutic molecules into deeper spheroid regions.

GSH-responsive SN38 dimer-loaded shape-transformable nanoparticles with iRGD for enhancing chemo-photodynamic therapy

Accurate tumor targeting, deep penetration and superb retention are still the main pursuit of developing excellent nanomedicine. To achieve these requirements, a stepwise stimuli-responsive strategy was developed through co-administration tumor penetration peptide iRGD with shape-transformable and GSH-responsive SN38-dimer (d-SN38)-loaded nanoparticles (d-SN38@NPs/iRGD). Upon intravenous injection, d-SN38@NPs with high drug loading efficiency (33.92 ± 1.33%) could effectively accumulate and penetrate into the deep region of tumor sites with the assistance of iRGD. The gathered nanoparticles simultaneously transformed into nanofibers upon 650 nm laser irradiation at tumor sites so as to promote their retention in the tumor and burst release of reactive oxygen species for photodynamic therapy. The loaded d-SN38 with disulfide bond responded to the high level of GSH in tumor cytoplasm, which consequently resulted in SN38 release and excellent chemo-photodynamic effect on tumor. In vitro, co-administering iRGD with d-SN38@NPs+laser showed higher cellular uptake, apoptosis ratio and multicellular spheroid penetration. In vivo, d-SN38@NPs/iRGD+laser displayed advanced penetration and accumulation in tumor, leading to 60.89% of tumor suppression in 4T1 tumor-bearing mouse model with a favorable toxicity profile. Our new strategy combining iRGD with structural transformable nanoparticles greatly improves tumor targeting, penetrating and retention, and empowers anticancer efficacy.

Oleanolic acid-conjugated poly (D, l-lactide)-based micelles for effective delivery of doxorubicin and combination chemotherapy in oral cancer

Here, an amphiphilic polymer, oleanolic acid conjugated methoxy-poly (ethylene glycol)-poly (D, l-lactide) (mPEG-PLA-OA) was synthesized, which self-assembled in aqueous environment and loaded the poorly soluble anticancer agent, doxorubicin. Various physico-chemical characterization studies on polymer and DOX-loaded polymeric micelles (DOX-mPEG-PLA-OA) at various drug to polymer (1:5/10/15) (DTP) ratios were performed, which included characterizations via UV, IR, NMR spectroscopy and gel permeation chromatography for polymers, and dynamic light scattering, DSC, XRD techniques, determination of critical micelles concentration, percent drug loading, encapsulation efficiency, drug release, kinetic stability and haemolysis studies for micelles. Further, DOX-mPEG-PLA-OA were evaluated for their cellular uptake, cytotoxicity, ability to induce apoptosis, DNA fragmentation, nuclear damage, ROS generation, and mitochondrial membrane depolarization in oral squamous cell carcinoma (FaDu HTB-43) and murine melanoma (B16F10) in monolayer and in FaDu HTB-43 3D cell culture systems. DOX-mPEG-PLA-OA at DTP ratios. 1:5/10/15 showed physico-chemical parameters in the following range: particle sizes. ~210.3–225.1 nm, DOX encapsulation efficiency. 29–35% and DOX-loading. 2.2–5%. DOX-mPEG-PLA-OA micelles (1:15) were stable as proven by kinetic stability study, and showed sustained release of encapsulated DOX for over 60 h. The cell viability study revealed that DOX-mPEG-PLA-OA micelles (1:15) demonstrated highest time- and dose dependent cytotoxicity compared to free DOX and micelles of other DTP ratios. Following treatment, the DOX-mPEG-PLA-OA micelles (1:5/10/15) induced highest cellular apoptosis in the range of 28.17–48.1, compared to free DOX (9.5 ± 2.52), and mPEG-PLA-OA micelles (25.4 ± 2.91), DOX-mPEG-PLA-OA micelles (1:15) being the strongest apoptosis inducer (48.1 ± 2.68) as determined by flow cytometry-mediated annexin V assay. DOX-mPEG-PLA-OA micelles (1:15) induced significantly higher DNA fragmentation, nuclear condensation, ROS generation, and mitochondrial membrane permeabilization compared to free Dox in monolayer FaDu HTB-43. DOX-mPEG-PLA-OA micelles (1:15) showed reasonably higher penetration and stronger growth inhibition in FaDu HTB-43 spheroids compared to free DOX and micelles of other DTP ratios. In-vivo pharmacokinetic studies showed that the DOX-mPEG-PLA-OA micelles demonstrated enhanced blood circulation time with decreased rate of clearance than free Dox (t1/2. 33.5 ± 2.5 h vs. 1.77 ± 0.32 h, and Cmax. 59.7 ± 2.2 vs. 2.3 ± 0.1, respectively). Immunohistochemistry revealed no significant toxicity in major organs of DOX-mPEG-PLA-OA micelles treated rats. The series of studies indicated that the OA-conjugated mPEG-PLA micelles served as an efficient nanocarriers for DOX, demonstrated DOX, and OA combined superior anticancer effect, sustained DOX level in blood circulation for up to 96 h, and no sign of major organs toxicity. Therefore, this newly developed DOX, OA combined micelles displayed its potential as promising alternative to DOX-based conventional therapy, and warrants further investigation for clinical application, especially for the treatment of oral cancer.

Cyclodextrin nanosponge as a temoporfin nanocarrier: Balancing between accumulation and penetration in 3D tumor spheroids

As the intertissue delivery of hydrophobic temoporfin (mTHPC) remains inefficient, we propose the use of cyclodextrin-based nanosponges as a smart, advanced system for improved mTHPC delivery. Recently, we demonstrated that cyclodextrins (CDs) allow mTHPC to penetrate into tumor spheroids via a nanoshuttle mechanism. However, the CD complexes were very sensitive to the dilution, thus limiting their translation invivo. Hypercrosslinked CD monomers in a three-dimensional network (namely, CD nanosponges), however, may form both inclusion and non-inclusion complexes with drug molecules, providing controlled release and prolonged exposure to the drug. In the present work, we demonstrate that epichlorohydrin-crosslinked CD nanosponges based on β-CD (βCDp) and carboxymethyl-β-CD (CMβCDp) monomers efficiently encapsulated mTHPC. We calculated the apparent binding constants between mTHPC and CD polymers (K=(6.3–8.8) × 106M−1 and K=(1.2–1.7) × 106M−1 for βCDp and CMβCDp, respectively) using fluorescence titration curve fitting. The encapsulation of mTHPC in a CD polymer matrix had slower photosensitizer (PS) release compared to monomer CD units, providing deep penetration of mTHPC in 3D tumor spheroids in a concentration-dependent manner. However, the improvement of mTHPC penetration in 3D human pharynx squamous cell carcinoma (FaDu) spheroids using CD polymers was strongly accompanied by the inhibition of PS cellular uptake, demonstrating the delicate balance between the accumulation and the penetration of PS in FaDu spheroids. In summary, mTHPC-loaded CD nanosponges are a strong candidate for further invivo study in preclinical models, which could be considered as an advanced smart system for mTHPC delivery.

RGD-decorated solid lipid nanoparticles enhance tumor targeting, penetration and anticancer effect of asiatic acid.

Aim: Asiatic acid (AA) is a promising anticancer agent, however, its delivery to glioblastoma is a major challenge. This work investigates the beneficial therapeutic efficacy of RGD-conjugated solid lipid nanoparticles (RGD-SLNs) for the selective targeting of AA to gliblastoma. Materials &methods: AA-containing RGD-SLNs were prepared using two different PEG-linker size. Targetability and efficacy were tested using monolayer cells and spheroid tumor models. Results: RGD-SLNs significantly improved cytotoxicity of AA against U87-MG monolayer cells and enhanced cellular uptake compared with non-RGD-containing SLNs. In spheroid models, AA-containing RGD-SLNs showed superior control in tumor growth, improved cytotoxicity and enhanced spheroid penetration when compared with AA alone or non-RGD-containing SLNs. Conclusion: This study illustrates the potential of AA-loaded RGD-SLNs as efficacious target-specific treatment for glioblastoma.

Chemo-Photothermal Combination Cancer Therapy with ROS Scavenging, Extracellular Matrix Depletion, and Tumor Immune Activation by Telmisartan and Diselenide-Paclitaxel Prodrug Loaded Nanoparticles.

Extracellular matrix (ECM) accumulating in the tumor microenvironment (TME) is generated by tumor-associated fibroblasts. It can elevate interstitial fluid pressure and form dense barriers in tumor tissues. Consequently, nanocarriers are hindered from permeating into deeper tumor sites. Thus, the programmed drug-releasing nanoparticles, G(TM)PPSP, were developed for TME remodeling and breast cancer therapy. Gelatin nanoparticles were linked with platinum nanoparticles (PtNPs) to obtain G(TM)PPSP with a size of 214.0 ± 5.0 nm. Telmisartan (TM) was loaded in gelatin nanoparticles. Paclitaxel (PTX) was attached to PtNPs via a dual redox responsive diselenide bond. TM release was mediated by MMP-2 because of gelatin degradation in TME, and then intracellular PTX was released because of diselenide linkage fracture triggered by ROS or glutathione. ECM was depleted owing to TGF-β downregulation by TM and direct ablation by the photothermal effect of PtNPs. 4T1 tumor progression was inhibited by PTX chemotherapy, intracellular ROS scavenging of PtNPs, and photothermal therapy (PTT). The tumor spheroid penetration assay proved G(TM)PPSP could permeate into deep tumor regions when MMP-2 existed. In vivo antitumor experiments implied G(TM)PPSP with PTT could inhibit tumor growth effectively and remodel TME via ECM depletion and immunity activation, indicating the potential of G(TM)PPSP-based chemo-photothermal combination therapy for breast cancer treatment.

Systematic evaluation of design features enables efficient selection of Π electron-stabilized polymeric micelles.

Polymeric micelles (PM) based on poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) loaded with paclitaxel (PTX-PM) have shown promising results in overcoming the suboptimal efficacy/toxicity profile of paclitaxel. To get insight into the stability of PTX-PM formulations upon storage and to optimize their in vivo tumor-targeted drug delivery properties, we set out to identify a lead PTX-PM formulation with the optimal polymer composition. To this end, PM based on four different mPEG5k-b-p(HPMA-Bz) block copolymers with varying molecular weight of the hydrophobic block (17-3kDa) were loaded with different amounts of PTX. The hydrodynamic diameter was 52±1nm for PM prepared using polymers with longer hydrophobic blocks (mPEG5k-b-p(HPMA-Bz)17k and mPEG5k-b-p(HPMA-Bz)10k) and 39±1nm for PM composed of polymers with shorter hydrophobic blocks (mPEG5k-b-p(HPMA-Bz)5k and mPEG5k-b-p(HPMA-Bz)3k). The best storage stability and the slowest PTX release was observed for PM with larger hydrophobic blocks. On the other hand, smaller sized PM of shorter mPEG5k-b-p(HPMA-Bz)5k showed a better tumor penetration in 3D spheroids. Considering better drug retention capacity of the mPEG5k-b-p(HPMA-Bz)17k and smaller size of the mPEG5k-b-p(HPMA-Bz)5k as two desirable design features, we argue that PM based on these two polymers are the lead candidates for further in vivo studies.

Preparation of Frozen Sections of Multicellular Tumor Spheroids Coated with Ice for Mass Spectrometry Imaging.

Increasing studies have utilized mass spectrometry imaging (MSI) that is a label-free tool to investigate drug penetration and drug biotransformation in multicellular tumor spheroids (MCTS). Currently, the gelatin-assisted sectioning method is widely used to prepare frozen sections of MCTS for MSI. However, owing to the limited transparency of frozen gelatin, MCTS with diameters less than 500 μm that closely mimic solid tumors are difficult to be detected when cryosectioning. In order to identify the presence of MCTS, hematoxylin and eosin staining for frozen sections and dye pretreatment for MCTS were employed in previous works, which either increased the analytical time and cost in sample preparation or caused signal suppression in sample analysis. Herein, a new sectioning method was developed to prepare MCTS frozen sections. MCTS was coated with ice to ensure good visibility for small-size MCTS. The optimal cutting temperature compound was added around the ice block to assist the formation of frozen sections. A precast frozen mold was prepared to allow the acquisition of complete MCTS frozen sections. The developed method was applied to investigate lipid distribution in MCTS by using matrix-assisted laser desorption/ionization MSI. Compared to the gelatin-assisted sectioning method, our method did not cause signal suppression and analyte delocalization. Thus, this method provides an easy, universal, and innovative strategy to prepare MCTS frozen sections for further MSI analysis. Besides, we applied our method to investigate the penetration of bisphenol A in MCTS.