Ex Vivo and In Vivo Evaluation of Dodecaborate-Based Clusters Encapsulated in Ferumoxytol Nanoparticles.

Host-guest interactions represent a growing research area with recent work demonstrating the ability to chemically manipulate both host molecules as well as guest molecules to vary the type and strength of bonding. Much less is known about the interactions of the guest molecules and hybrid materials containing similar chemical features to typical macrocyclic hosts. This work uses in vitro and in vivo kinetic analyses to investigate the interaction of closo-dodecahydrododecaborate derivatives with ferumoxytol, an iron oxide nanoparticle with a carboxylated dextran coating. We find that several boron cluster derivatives can become encapsulated into ferumoxytol, and the lack of pH dependence in these interactions suggests that ion pairing, hydrophobic/hydrophilic interaction, and hydrogen bonding are not the driving force for encapsulation in this system. Biodistribution experiments in BALB/c mice show that this system is nontoxic at the reported dosage and demonstrate that encapsulation of dodecaborate-based clusters in ferumoxytol can alter the biodistribution of the guest molecules.

Intraoperative assessment and postsurgical treatment of prostate cancer tumors using tumor-targeted nanoprobes.

Successful visualization of prostate cancer (PCa) tumor margins during surgery remains a major challenge. The visualization of these tumors during surgery via near infrared fluorescence (NIRF) imaging would greatly enhance surgical resection, minimizing tumor recurrence and improving outcome. Furthermore, chemotherapy is typically administered to patients after surgery to treat any missed tumor tissue around the surgical area, minimizing metastasis and increasing patient survival. For these reasons, a theranostics fluorescent nanoparticle could be developed to assist in the visualization of PCa tumor margins, while also delivering chemotherapeutic drug after surgery. Methods: Ferumoxytol (FMX) conjugated to the fluorescent dye and PCa targeting agent, heptamethine carbocyanine (HMC), yielded the HMC-FMX nanoprobe that was tested in vitro with various PCa cell lines and in vivo with both subcutaneous and orthotopic PCa mouse models. Visualization of these tumors via NIRF imaging after administration of HMC-FMX was performed. In addition, delivery of chemotherapeutic drug and their effect on tumor growth was also assessed. Results: HMC-FMX internalized into PCa cells, labeling these cells and PCa tumors in mice with near infrared fluorescence, facilitating tumor margin visualization. HMC-FMX was also able to deliver drugs to these tumors, reducing cell migration and slowing down tumor growth. Conclusion: HMC-FMX specifically targeted PCa tumors in mice allowing for the visualization of tumor margins by NIRF imaging. Furthermore, delivery of anticancer drugs by HMC-FMX effectively reduced prostate tumor growth and reduced cell migration in vitro. Thus, HMC-FMX can potentially translate into the clinic as a nanotheranostics agent for the intraoperative visualization of PCa tumor margins, and post-operative treatment of tumors with HMC-FMX loaded with anticancer drugs.

A Translational Porcine Model for Human Cell-Based Therapies in the Treatment of Posttraumatic Osteoarthritis After Anterior Cruciate Ligament Injury.

BACKGROUND: There is a high incidence of posttraumatic osteoarthritis (PTOA) after anterior cruciate ligament (ACL) injury, and these injuries represent an enormous health care economic burden. In an effort to address this unmet clinical need, there has been increasing interest in cell-based therapies. PURPOSE: To establish a translational large animal model of PTOA and demonstrate the feasibility of intra-articular human cell-based interventions. STUDY DESIGN: Descriptive laboratory study. METHODS: Nine Yucatan mini-pigs underwent unilateral ACL transection and were monitored for up to 12 weeks after injury. Interleukin 1 beta (IL-1ß) levels and collagen breakdown were evaluated longitudinally using enzyme-linked immunosorbent assays of synovial fluid, serum, and urine. Animals were euthanized at 4 weeks (n = 3) or 12 weeks (n = 3) after injury, and injured and uninjured limbs underwent magnetic resonance imaging (MRI) and histologic analysis. At 2 days after ACL injury, an additional 3 animals received an intra-articular injection of 107 human bone marrow-derived mesenchymal stem cells (hBM-MSCs) combined with a fibrin carrier. These cells were labeled with the luciferase reporter gene (hBM-MSCs-Luc) as well as fluorescent markers and intracellular iron nanoparticles. These animals were euthanized on day 0 (n = 1) or day 14 (n = 2) after injection. hBM-MSC-Luc viability and localization were assessed using ex vivo bioluminescence imaging, fluorescence imaging, and MRI. RESULTS: PTOA was detected as early as 4 weeks after injury. At 12 weeks after injury, osteoarthritis could be detected grossly as well as on histologic analysis. Synovial fluid analysis showed elevation of IL-1ß shortly after ACL injury, with subsequent resolution by 2 weeks after injury. Collagen type II protein fragments were elevated in the synovial fluid and serum after injury. hBM-MSCs-Luc were detected immediately after injection and at 2 weeks after injection using fluorescence imaging, MRI, and bioluminescence imaging. CONCLUSION: This study demonstrates the feasibility of reproducing the chondral changes, intra-articular cytokine alterations, and body fluid biomarker findings consistent with PTOA after ACL injury in a large animal model. Furthermore, we have demonstrated the ability of hBM-MSCs to survive and express transgene within the knee joint of porcine hosts without immunosuppression for at least 2 weeks. CLINICAL RELEVANCE: This model holds great potential to significantly contribute to investigations focused on the development of cell-based therapies for human ACL injury-associated PTOA in the future (see Appendix Figure A1, available online).

Near Infrared Fluorescent Nanoplatform for Targeted Intraoperative Resection and Chemotherapeutic Treatment of Glioblastoma.

Despite significant efforts to improve glioblastoma multiforme (GBM) treatment, GBM remains one of the most lethal cancers. Effective GBM treatments require sensitive intraoperative tumor visualization and effective postoperative chemotherapeutic delivery. Unfortunately, the diffusive and infiltrating nature of GBM limits the detection of GBM tumors, and current intraoperative visualization methods limit complete tumor resection. In addition, although chemotherapy is often used to eliminate any cancerous tissue remaining after surgery, most chemotherapeutic drugs do not effectively cross the brain-blood barrier (BBB) or enter GBM tumors. As a result, GBM has limited treatment options with high recurrence rates, and methods that improve its complete visualization during surgery and treatment are needed. Herein, we report a fluorescent nanoparticle platform for the near-infrared fluorescence (NIRF)-based tumor boundary visualization and image-guided drug delivery into GBM tumors. Our nanoplatform is based on ferumoxytol (FMX), an FDA-approved magnetic resonance imaging-sensitive superparamagnetic iron oxide nanoparticle, which is conjugated with hepthamethine cyanine (HMC), a NIRF ligand that specifically targets the organic anion transporter polypeptides that are overexpressed in GBM. We have shown that HMC-FMX nanoparticles cross the BBB and selectively accumulate in the tumor using orthotopic GBM mouse models, enabling NIRF-based visualization of infiltrating tumor tissue. In addition, HMC-FMX can encapsulate chemotherapeutic drugs, such as paclitaxel or cisplatin, and deliver these agents into GBM tumors, reducing tumor size and increasing survival. Taken together, these observations indicate that HMC-FMX is a promising nanoprobe for GBM surgical visualization and drug delivery.

Tumor-Activatable Clinical Nanoprobe for Cancer Imaging.

Purpose: A successful cancer surgery requires the complete removal of cancerous tissue, while also sparing as much healthy, non-cancerous tissue as possible. To achieve this, an accurate identification of tumor boundaries during surgery is critical, but intra-operative tumor visualization remains challenging. Fluorescence imaging is a promising method to improve tumor detection and delineate tumor boundaries during surgery, but the lack of stable, long-circulating, clinically-translatable fluorescent probes that can identify tumors with high signal-to-noise ratios and low background fluorescence signals have prevented its widespread application. Methods: We screened the optical properties of several fluorescent dyes before and after nanoprobe encapsulation, and then identified nanoprobes with quenched fluorescence that were re-activated upon dye release. The physical and biological properties of these nanoprobes leading to fluorescence activation were investigated in vitro. Further, the cancer imaging properties of both free dyes and nanoprobe-encapsulated dyes were compared in vivo. Results: A novel fluorescent nanoprobe was prepared by combining two FDA-approved agents commonly used in the clinic: Feraheme (FH) and indocyanine green (ICG). The resulting FH-entrapped ICG nanoprobe [FH(ICG)] displayed quenched fluorescence compared to other nanoprobes, and this quenched fluorescence was re-activated in acidic tumor microenvironment conditions (pH 6.8) and upon uptake into cancer cells. Finally, in vivo studies in a prostate cancer mouse model demonstrated that FH(ICG) treatments enhance long-term fluorescence signals in tumors compared to ICG treatments, allowing for fluorescence-guided tumor identification using clinically relevant fluorescence cameras. Conclusions: FH(ICG) nanoprobes were identified as fluorescent nanoprobes with beneficial fluorescence activation properties compared to other FH-entrapped dyes. The activatable nature of this nanoprobe allows for a low background fluorescence signal and high signal-to-noise ratio within a long-circulating nanoagent, which allows for long-term fluorescence signals from tumors that enabled their fluorescence-guided detection. This activatable nanoprobe offers tremendous potential as a clinically translatable image-guided cancer therapy modality that can be prepared in a clinical setting.

Biological Effects of Nanoparticles on Macrophage Polarization in the Tumor Microenvironment.

Biological interactions between tumor-associated macrophages (TAMs), cancer cells and other cells within the tumor microenvironment contribute to tumorigenesis, tumor growth, metastasis and therapeutic resistance. TAMs can remodel the tumor microenvironment to reduce growth barriers such as the dense extracellular matrix and shift tumors towards an immunosuppressive microenvironment that protects cancer cells from targeted immune responses. Nanoparticles can interrupt these biological interactions within tumors by altering TAM phenotypes through a process called polarization. Macrophage polarization within tumors can shift TAMs from a growth-promoting phenotype towards a cancer cell-killing phenotype that predicts treatment efficacy. Because many types of nanoparticles have been shown to preferentially accumulate within macrophages following systemic administration, there is considerable interest in identifying nanoparticle effects on TAM polarization, evaluating nanoparticle-induced TAM polarization effects on cancer treatment using drug-loaded nanoparticles and identifying beneficial types of nanoparticles for effective cancer treatment. In this review, the macrophage polarization effects of nanoparticles will be described based on their primary chemical composition. Because of their strong macrophage-polarizing and antitumor effects compared to other types of nanoparticles, the effects of iron oxide nanoparticles on macrophages will be discussed in detail. By comparing the macrophage polarization effects of various nanoparticle treatments reported in the literature, this review aims to both elucidate nanoparticle material effects on macrophage polarization and to provide insight into engineering nanoparticles with more beneficial immunological responses for cancer treatment.

In vivo ß-catenin attenuation by the integrin α5-targeting nano-delivery strategy suppresses triple negative breast cancer stemness and metastasis.

Cancer stem cells (CSCs) play pivotal roles in cancer metastasis, and strategies targeting cancer stemness may greatly reduce cancer metastasis and improve patients' survival. The canonical Wnt/ß-catenin pathway plays critical roles in CSC generation and maintenance as well as in normal stem cells. Non-specifically suppressing the Wnt/ß-catenin pathway for cancer therapy could be deleterious to normal cells. To achieve specific ß-catenin attenuation in cancer cells, we report an integrin α5 (ITGA5)-targeting nanoparticle for treating metastatic triple negative breast cancer (TNBC). We found that ITGA5 is highly expressed in strongly migratory and invasive TNBC cells as well as their lung metastatic foci, which rationalizes active-targeted drug delivery to TNBC cells via ITGA5 ligands such as a commercialized ligand-RGD motif (Arg-Gly-Asp). We modified lipid-polymer hybrid (LPH) nanoparticle for TNBC-targeted delivery of diacidic norcantharidin (NCTD), a potent anti-cancer compound but with short half-life. Notably, in vivo imaging analysis showed that RGD-decorated LPH (RGD-LPH) accumulated more significantly and remained much longer than LPH in nude mouse orthotopic mammary TNBC tumor and lung metastatic tumor, which implicated the feasibility of ITGA5-targeting strategy for treating metastatic TNBC. Moreover, systemic administration of NCTD-loaded RGD-LPH (RGD-LPH-NCTD) reduced nude mouse orthotopic mammary TNBC tumor growth and metastasis more effectively than free NCTD and LPH-NCTD via down-regulating ß-catenin. These findings suggest that ITGA5-targeting nanoparticles may provide a facil and unique strategy of specially attenuating ß-catenin in vivo for treating metastatic TNBC.

Colorectal cancer lung metastasis treatment with polymer-drug nanoparticles.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States; the predominant cause for mortality is metastasis to distant organs (e.g., lung). A major problem limiting the success of chemotherapy in metastatic CRC is the inability to target tumor tissues selectively and avoid severe side effects to normal tissues and organs. Here, we demonstrate polymeric nanoparticles (PNPs) entrapping chemotherapeutic agents provide a new therapeutic option for treating CRC that has metastasized to the lung. PNPs assembled from FDA approved biocompatible block copolymer accumulated predominantly in lung tissue. PNPs showed negligible accumulation in liver, spleen and kidneys, which was confirmed by fluorescent nanoparticle imaging and analysis of PI3K inhibition in the organs. PNPs entrapping PI3K inhibitors (i.e., wortmannin and PX866) suppressed CRC lung metastasis growth, and SN-38-loaded PNPs completely eliminated CRC lung metastasis. Our results demonstrate that polymer-drug nanoparticles offer a new approach to reduce toxicity of cancer therapy and has the potential to improve outcomes for patients with lung metastasis.

Leukemia Inhibitory Factor-Loaded Nanoparticles with Enhanced Cytokine Metabolic Stability and Anti-Inflammatory Activity.

PURPOSE: To synthesize and assess the in vitro biological activity of nanoparticles containing leukemia inhibitory factor (LIF). These NanoLIF particles are designed to prolong the neuroprotective and anti-inflammatory actions of LIF in future preclinical studies of ischemic stroke. METHODS: LIF was packaged in nanoparticles made of poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) polymer to form LIF-loaded nanoparticles (NanoLIF). The surface of NanoLIF was also modified with the CD11b antibody (CD11b-NanoLIF) targeting activated peripheral macrophages to increase cytokine delivery to inflammatory macrophages. ELISA was used to quantify bioactive cytokine inside and releasing from NanoLIF. NanoLIF biological activity was measured using the M1 murine leukemia cell proliferation assay. RESULTS: NanoLIF and CD11b-NanoLIF had diameters of approximately 30 nm, neutral surface charge, and physicochemical stability retaining biological activity of the cytokine during incubation at 25°C for 12 h. NanoLIF particles released LIF relatively fast from 0 to 6 h after incubation at 37°C followed by slow release from 24 to 72 h according to a two-phase exponential decay model. NanoLIF and CD11b-NanoLIF significantly decreased M1 cell proliferation over 72 h compared to free LIF. CONCLUSIONS: NanoLIF and CD11b-NanoLIF preserved the metabolic stability and biological activity of LIF in vitro. These results are promising to improve the therapeutic potential of LIF in treating neurodegenerative and inflammatory diseases.

Development of Halofluorochromic Polymer Nanoassemblies for the Potential Detection of Liver Metastatic Colorectal Cancer Tumors Using Experimental and Computational Approaches.

PURPOSE: To develop polymer nanoassemblies (PNAs) modified with halofluorochromic dyes to allow for the detection of liver metastatic colorectal cancer (CRC) to improve therapeutic outcomes. METHODS: We combine experimental and computational approaches to evaluate macroscopic and microscopic PNA distributions in patient-derived xenograft primary and orthotropic liver metastatic CRC tumors. Halofluorochromic and non-halofluorochromic PNAs (hfPNAs and n-hfPNAs) were prepared from poly(ethylene glycol), fluorescent dyes (Nile blue, Alexa546, and IR820), and hydrophobic groups (palmitate), all of which were covalently tethered to a cationic polymer scaffold [poly(ethylene imine) or poly(lysine)] forming particles with an average diameter < 30 nm. RESULTS: Dye-conjugated PNAs showed no aggregation under opsonizing conditions for 24 h and displayed low tissue diffusion and cellular uptake. Both hfPNAs and n-hfPNAs accumulated in primary and liver metastatic CRC tumors within 12 h post intravenous injection. In comparison to n-hfPNAs, hfPNAs fluoresced strongly only in the acidic tumor microenvironment (pH < 7.0) and distinguished small metastatic CRC tumors from healthy liver stroma. Computational simulations revealed that PNAs would steadily accumulate mainly in acidic (hypoxic) interstitium of metastatic tumors, independently of the vascularization degree of the tissue surrounding the lesions. CONCLUSION: The combined experimental and computational data confirms that hfPNAs detecting acidic tumor tissue can be used to identify small liver metastatic CRC tumors with improved accuracy.

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays.

Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(l-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P' caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'<2PD<3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay.

Polymer micelle formulation for the proteasome inhibitor drug carfilzomib: Anticancer efficacy and pharmacokinetic studies in mice.

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor approved for the treatment of multiple myeloma (MM). Despite the remarkable efficacy of CFZ against MM, the clinical trials in patients with solid cancers yielded rather disappointing results with minimal clinical benefits. Rapid degradation of CFZ in vivo and its poor penetration to tumor sites are considered to be major factors limiting its efficacy against solid cancers. We previously reported that polymer micelles (PMs) composed of biodegradable block copolymers poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) can improve the metabolic stability of CFZ in vitro. Here, we prepared the CFZ-loaded PM, PEG-PCL-deoxycholic acid (CFZ-PM) and assessed its in vivo anticancer efficacy and pharmacokinetic profiles. Despite in vitro metabolic protection of CFZ, CFZ-PM did not display in vivo anticancer efficacy in mice bearing human lung cancer xenograft (H460) superior to that of the clinically used cyclodextrin-based CFZ (CFZ-CD) formulation. The plasma pharmacokinetic profiles of CFZ-PM were also comparable to those of CFZ-CD and the residual tumors that persisted in xenograft mice receiving CFZ-PM displayed an incomplete proteasome inhibition. In summary, our results showed that despite its favorable in vitro performances, the current CFZ-PM formulation did not improve in vivo anticancer efficacy and accessibility of active CFZ to solid cancer tissues over CFZ-CD. Careful consideration of the current results and potential confounding factors may provide valuable insights into the future efforts to validate the potential of CFZ-based therapy for solid cancer and to develop effective CFZ delivery strategies that can be used to treat solid cancers.

Comparison of Dialysis- and Solvatofluorochromism-Based Methods to Determine Drug Release Rates from Polymer Nanoassemblies.

PURPOSE: To compare traditional dialysis- and novel solvatofluorochromism (SFC)-based methods for accurate determination of drug release profiles for nanoparticle drug carriers. METHODS: Polymer nanoassemblies (PNAs) varying in drug release patterns were prepared using poly(ethylene glycol), poly(ethylenimine), hydrophobic excipients (palmitate and deoxycholate), and model hydrophobic anticancer drugs with clinical relevance (carfilzomib and docetaxel). Nile blue (NB) was used as a model SFC dye quenching fluorescence in water yet emitting strong fluorescence in the presence of hydrophobic drugs within PNAs. Drug release kinetics were measured by dialysis- and SFC-based methods, and analyzed by mathematical modeling of free drug, spiked drug, and encapsulated drug release. RESULTS: The dialysis method overestimated drug remaining in PNAs because it included released drug in measurements, whereas the SFC method successfully distinguished drugs entrapped in PNAs from released in solution and thus provided more accurate drug release patterns. However, mathematical modeling revealed that the dialysis method would be less influenced than the SFC method by hydrophobic excipients modulating drug diffusion within PNAs. CONCLUSIONS: In comparison to the dialysis-based method, the SFC-based method would allow for real-time spectroscopic determination of drug release from PNAs and potentially other nanoparticle drug carriers with improved convenience and accuracy.

Tethered polymer nanoassemblies for sustained carfilzomib release and prolonged suppression of proteasome activity.

AIM: Proteasome inhibitors, such as carfilzomib (CFZ), have shown potential to treat various types of cancers in preclinical models, but clinical applications are limited likely due to formulation and delivery issues. Results & methodology: Tethered polymer nanoassemblies (TNAs) were synthesized by tethering hydrophilic polymers and hydrophobic groups to charged polymer scaffolds, and then end-capping remaining amines on scaffold. Drug entrapment and drug release half-lives increased as charge was removed from scaffold. TNAs with sustained CFZ release maintained drug efficacy after preincubation and increased duration of proteasome inhibition in cancer cells compared with free CFZ. CONCLUSION: TNAs fine-tuned CFZ release as charge was removed from polymer scaffold, which allowed for sustained proteasome inhibition in cancer cells and potentially enhanced anticancer efficacy.

Polymer nanoassemblies with solvato- and halo-fluorochromism for drug release monitoring and metastasis imaging.

BACKGROUND: Theranostics, an emerging technique that combines therapeutic and diagnostic modalities for various diseases, holds promise to detect cancer in early stages, eradicate metastatic tumors and ultimately reduce cancer mortality. METHODS & RESULTS: This study reports unique polymer nanoassemblies that increase fluorescence intensity upon addition of hydrophobic drugs and either increase or decrease fluorescence intensity in acidic environments, depending on nanoparticle core environment properties. Extensive spectroscopic analyses were performed to determine optimal excitation and emission wavelengths, which enabled real time measurement of drugs releasing from the nanoassemblies and ex vivo imaging of acidic liver metastatic tumors from mice. CONCLUSION: Polymer nanoassemblies with solvato- and halo-fluorochromic properties are promising platforms to develop novel theranostic tools for the detection and treatment of metastatic tumors.

Polymer micelle formulations of proteasome inhibitor carfilzomib for improved metabolic stability and anticancer efficacy in human multiple myeloma and lung cancer cell lines.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor drug approved for the treatment of multiple myeloma. Contrary to its excellent antimyeloma activity, CFZ has shown only limited efficacy in patients with solid malignancies. This lack of efficacy has been attributed in part to rapid degradation of CFZ in the body, possibly hindering the ability of CFZ to access the proteasome target in solid tumors. We hypothesized that polymer micelles, a currently Food and Drug Administration-approved nanoparticle drug delivery formulation, may protect CFZ from metabolic degradation and thus expand the clinical utility of the drug as an anticancer agent. To test our hypothesis, we prepared CFZ-entrapped polymer micelle particles with various compositions and drug release profiles and examined the extent of the CFZ metabolism in vitro using mouse liver homogenates. We also assessed the cytotoxic activities of the CFZ-entrapped micelle formulations in human cancer cell lines derived from B lymphocytes (RPMI-8226) and the lung (H460). Our data indicated that polymer micelle-based formulations can improve metabolic stability and cytotoxic effects of CFZ compared with free CFZ in human cancer cell lines tested. Taken together, these results suggest that polymer micelles may have potential as a delivery system for CFZ with an extended therapeutic utility for nonhematologic malignancies in the future.