Preclinical Efficacy of Cabazitaxel Loaded Poly(2-alkyl cyanoacrylate) Nanoparticle Variants.

Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

Intraperitoneal administration of cabazitaxel-loaded nanoparticles in peritoneal metastasis models.

Colorectal and ovarian cancers frequently develop peritoneal metastases with few treatment options. Intraperitoneal chemotherapy has shown promising therapeutic effects, but is limited by rapid drug clearance and systemic toxicity. We therefore encapsulated the cabazitaxel taxane in poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs), designed to improve intraperitoneal delivery. Toxicity of free and encapsulated cabazitaxel was investigated in rats by monitoring clinical signs, organ weight and blood hematological and biochemical parameters. Pharmacokinetics, biodistribution and treatment response were evaluated in mice. Biodistribution was investigated by measuring both cabazitaxel and the 2-ethylbutanol NP degradation product. Drug encapsulation was shown to increase intraperitoneal drug retention, leading to prolonged intraperitoneal drug residence time and higher drug concentrations in peritoneal tumors. As a result, encapsulation of cabazitaxel improved the treatment response in two in vivo models bearing intraperitoneal tumors. Together, these observations indicate a strong therapeutic potential of NP-based cabazitaxel encapsulation as a novel treatment for peritoneal metastases.

Alginate Microsphere Encapsulation of Drug-Loaded Nanoparticles: A Novel Strategy for Intraperitoneal Drug Delivery.

Alginate hydrogels have been broadly investigated for use in medical applications due to their biocompatibility and the possibility to encapsulate cells, proteins, and drugs. In the treatment of peritoneal metastasis, rapid drug clearance from the peritoneal cavity is a major challenge. Aiming to delay drug absorption and reduce toxic side effects, cabazitaxel (CAB)-loaded poly(alkyl cyanoacrylate) (PACA) nanoparticles were encapsulated in alginate microspheres. The PACAlg alginate microspheres were synthesized by electrostatic droplet generation and the physicochemical properties, stability, drug release kinetics, and mesothelial cytotoxicity were analyzed before biodistribution and therapeutic efficacy were studied in mice. The 450 µm microspheres were stable at in vivo conditions for at least 21 days after intraperitoneal implantation in mice, and distributed evenly throughout the peritoneal cavity without aggregation or adhesion. The nanoparticles were stably retained in the alginate microspheres, and nanoparticle toxicity to mesothelial cells was reduced, while the therapeutic efficacy of free CAB was maintained or improved in vivo. Altogether, this work presents the alginate encapsulation of drug-loaded nanoparticles as a promising novel strategy for the treatment of peritoneal metastasis that can improve the therapeutic ratio between toxicity and therapeutic efficacy.

High-throughput assay for effect screening of amphotericin B and bioactive components on filamentous Candida albicans.

AIMS: The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB). METHODS AND RESULTS: Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 µg ml-1 , and this was eightfold reduced (0.0038 µg ml-1 ) in the presence of the nutritional drinks. CONCLUSIONS: The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development.

Identification of novel cyanoacrylate monomers for use in nanoparticle drug delivery systems prepared by miniemulsion polymerisation - A multistep screening approach.

Poly (alkyl cyanoacrylate) (PACA) polymeric nanoparticles (NPs) are promising drug carriers in drug delivery. However, the selection of commercially available alkyl cyanoacrylate (ACA) monomers is limited, because most monomers were designed for use in medical and industrial glues and later repurposed for drug encapsulation. This study therefore aimed to seek out novel ACA materials for use in NP systems using a toxicity led screening approach. A multistep strategy, including cytotoxicity screening of alcohols as degradation products of PACA (44 alcohols), NPs (14 polymers), and a final in vivo study (2 polymers) gave poly (2-ethylhexyl cyanoacrylate) PEHCA as a promising novel PACA candidate. For the first time, this work presents cytotoxicity data on several novel ACAs, PEHCA in vivo toxicity data, and miniemulsion polymerisation-based encapsulation of the cabazitaxel and NR688 in novel PACA candidates. Furthermore, several of the ACA candidates were compatible with a wider selection of lipophilic active pharmaceutical ingredients (APIs) versus commercially available controls. Combined, this work demonstrates the potential benefits of expanding the array of available ACA materials in drug delivery. Novel ACAs have the potential to encapsulate a wider range of APIs in miniemulsion polymerisation processes and may also broaden PACA applicability in other fields.

Interlaboratory evaluation of a digital holographic microscopy-based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers.

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.

A multistep in vitro hemocompatibility testing protocol recapitulating the foreign body reaction to nanocarriers.

The development of drug nanocarriers based on polymeric, lipid and ceramic biomaterials has been paving the way to precision medicine, where the delivery of poorly soluble active compounds and personalized doses are made possible. However, the nano-size character of these carriers has been demonstrated to have the potential to elicit pathways of the host response different from those of the same biomaterials when engineered as larger size implants and of the drugs when administered without a carrier. Therefore, a specific regulatory framework needs to be made available that can offer robust scientific insights and provide safety data by reliable tests of these novel nano-devices. In this context, the present work presents a multistep protocol for the in vitro assessment of the hemocompatibility of nanocarriers of different physicochemical properties. Poly (ethyl butyl cyanoacrylate) nanoparticles and lipid-based (LipImage™ 815) nanoparticles of comparable hydrodynamic diameter were tested through a battery of assays using human peripheral blood samples and recapitulating the main pathways of the host response upon systemic administration; i.e., protein interactions, fibrinogen-platelet binding, cytotoxicity, and inflammatory response. The data showed the sensitivity and reproducibility of the methods adopted that were also demonstrated to determine individual variability as well as to discriminate between activation of pathways of inflammation and unintended release of inflammatory signaling caused by loss of cell integrity. Therefore, this multistep testing is proposed as a reliable protocol for nanoparticle development and emerging regulatory frameworks.

Subcellular detection of PEBCA particles in macrophages: combining darkfield microscopy, confocal Raman microscopy, and ToF-SIMS analysis.

The detection of biomedical organic nanocarriers in cells and tissues is still an experimental challenge. Here we developed an imaging strategy for the label-free detection of poly (ethylbutyl cyanoacrylate) (PEBCA) particles. Experiments were carried out with phagocytic NR8383 macrophages exposed to non-toxic and non-activating concentrations of fluorescent (PEBCA NR668 and PEBCA NR668/IR), non-fluorescent (PEBCA), and cabazitaxel-loaded PEBCA particles (PEBCA CBZ). Exposure to PEBCA NR668 revealed an inhomogeneous particle uptake similar to what was obtained with the free modified Nile Red dye (NR668). In order to successfully identify the PEBCA-loaded cells under label-free conditions, we developed an imaging strategy based on enhanced darkfield microscopy (DFM), followed by confocal Raman microscopy (CRM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Nitrile groups of the PEBCA matrix and PEBCA ions were used as suitable analytes for CRM and ToF-SIMS, respectively. Masses found with ToF-SIMS were further confirmed by Orbitrap-SIMS. The combined approach allowed to image small (< 1 µm) PEBCA-containing phagolysosomes, which were identified as PEBCA-containing compartments in NR8383 cells by electron microscopy. The combination of DFM, CRM, and ToF-SIMS is a promising strategy for the label-free detection of PEBCA particles.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles: toxicity and changes in the proteome of breast, colon and prostate cancer cells.

Nanoparticles composed of poly(alkyl cyanoacrylate) (PACA) have shown great promise due to their biodegradability and high drug loading capacity. Development of optimal PACA nanocarriers requires detailed analysis of the overall cellular impact exerted by PACA variants. We here perform a comprehensive comparison of cabazitaxel (CBZ)-loaded nanocarriers composed of three different PACA monomers, i.e. poly(n-butyl cyanoacrylate) (PBCA), poly(2-ethylbutyl cyanoacrylate) (PEBCA) and poly(octyl cyanoacrylate) (POCA). The cytotoxicity of drug-loaded and empty PACA nanoparticles were compared to that of free CBZ across a panel of nine cancer cell lines by assessing cellular metabolism, proliferation and protein synthesis. The analyses revealed that the cytotoxicity of all CBZ-loaded PACAs was similar to that of free CBZ for all cell lines tested, whereas the empty PACAs exerted much lower toxicity. To increase our understanding of the toxic effects of these treatments comprehensive MS-based proteomics were performed with HCT116, MDA-MB-231 and PC3 cells incubated with PACA-CBZ variants or free CBZ. Interestingly, PACA-CBZ specifically led to decreased levels of proteins involved in focal adhesion and stress fibers in all cell lines. Since we recently demonstrated that encapsulation of CBZ within PEBCA nanoparticles significantly improved the therapeutic effect of CBZ on a patient derived xenograft model in mice, we investigated the effects of this PACA variant more closely by immunoblotting. Interestingly, we detected several changes in the protein expression and degree of phosphorylation of SRC-pathway proteins that can be relevant for the therapeutic effects of these substances.

Sonoporation Using Nanoparticle-Loaded Microbubbles Increases Cellular Uptake of Nanoparticles Compared to Co-Incubation of Nanoparticles and Microbubbles.

Therapeutic agents can benefit from encapsulation in nanoparticles, due to improved pharmacokinetics and biodistribution, protection from degradation, increased cellular uptake and sustained release. Microbubbles in combination with ultrasound have been shown to improve the delivery of nanoparticles and drugs to tumors and across the blood-brain barrier. Here, we evaluate two different microbubbles for enhancing the delivery of polymeric nanoparticles to cells in vitro: a commercially available lipid microbubble (Sonazoid) and a microbubble with a shell composed of protein and nanoparticles. Various ultrasound parameters are applied and confocal microscopy is employed to image cellular uptake. Ultrasound enhanced cellular uptake depending on the pressure and duty cycle. The responsible mechanisms are probably sonoporation and sonoprinting, followed by uptake, and to a smaller degree enhanced endocytosis. The use of commercial Sonazoid microbubbles leads to significantly lower uptake than when using nanoparticle-loaded microbubbles, suggesting that proximity between cells, nanoparticles and microbubbles is important, and that mainly nanoparticles in the shell are taken up, rather than free nanoparticles in solution.

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft.

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

Sonopermeation Enhances Uptake and Therapeutic Effect of Free and Encapsulated Cabazitaxel.

Delivery of drugs and nanomedicines to tumors is often heterogeneous and insufficient and, thus, of limited efficacy. Microbubbles in combination with ultrasound have been found to improve delivery to tumors, enhancing accumulation and penetration. We used a subcutaneous prostate cancer xenograft model in mice to investigate the effect of free and nanoparticle-encapsulated cabazitaxel in combination with ultrasound and microbubbles with a lipid shell or a shell of nanoparticles. Sonopermeation reduced tumor growth and prolonged survival (26%-100%), whether the free drug was co-injected with lipid-shelled microbubbles or the nanoformulation was co-injected with lipid-shelled or nanoparticle-shelled microbubbles. Coherently with the improved therapeutic response, we found enhanced uptake of nanoparticles directly after ultrasound treatment that lasted several weeks (2.3 × -15.8 × increase). Neither cavitation dose nor total accumulation of nanoparticles could explain the variation within treatment groups, emphasizing the need for a better understanding of the tumor biology and mechanisms involved in ultrasound-mediated treatment.

Structural Variants of poly(alkylcyanoacrylate) Nanoparticles Differentially Affect LC3 and Autophagic Cargo Degradation.

Nanoparticle drug carriers trigger a variety of cellular stress responses, including ER stress and antioxidant responses, but may also affect the intracellular degradative pathway autophagy. This can impose profound effects on drug delivery, cellular treatment responses, and nanoparticle cytotoxicity. We recently demonstrated that even small variations in the alkyl side chains of poly(alkylcyanoacrylate) (PACA) drug carrier nanoparticles, namely butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), differentially induce ER stress and redox imbalance in human cell lines. Here, we systematically investigate how these PACA variants affect autophagy. Interestingly, treatment with PEBCA or POCA particles led to intracellular accumulation of the autophagosome marker LC3-II, but via different mechanisms. PEBCA induced an integrated stress response-and ATF4-mediated increase in LC3B mRNA, whereas POCA blocked autophagic degradation of LC3-II and long-lived proteins in bulk. PBCA also increased LC3B mRNA via the integrated stress response and ATF4, but unlike PEBCA, it inhibited LC3 lipidation and autophagic cargo degradation. Our data demonstrate that even subtle variations in NP structure can have profoundly different impacts on autophagy, and that careful monitoring of autophagic flux and cargo degradation is critical for drawing accurate conclusions. Our findings have important implications for the choice of PACA monomer in different therapeutic settings.

Experimental Treatment of Mucinous Peritoneal Metastases Using Patient-Derived Xenograft Models.

Mucinous peritoneal metastases (PM) generally respond poorly to systemic treatment, and there is a clear unmet need for new treatment strategies to improve survival and quality of life for patients with PM. In this work, the growth inhibitory effect of five drugs (oxaliplatin (OXA; 5 mg/kg), irinotecan (IRI; 60 mg/kg), cabazitaxel (CBZ; 15 or 30 mg/kg), regorafenib (REG; 10, 30 or 60 mg/kg), and capecitabine (CAP; 359 or 755 mg/kg) was investigated in three orthotopic patient-derived xenograft models that mimic mucinous PM. Drugs were administered intraperitoneally (i.p.) as monotherapy weekly for 4 weeks (OXA, IRI), as one single i.p. injection (CBZ), or orally (REG, CAP) daily 5 of 7 days per week for four weeks, and i.p. tumor growth and survival were monitored and compared between treatment groups. The i.p. administered drugs (OXA, IRI, CBZ) had the strongest growth inhibitory effect, with OXA being most efficacious, completely inhibiting tumor growth in the majority of the animals. CBZ and IRI also strongly inhibited tumor growth, but with more variation in efficacy between the models. A moderate reduction in tumor growth was observed in all models treated with REG, while CAP had little to no growth inhibitory effect. Targeted next-generation-sequencing has identified mutational profiles typically associated with PM (mutations in KRAS, GNAS, and BRAF oncogenes), supporting the representativeness of the models. The results presented in this work support the continued exploration of i.p. treatment protocols for PM, with OXA remaining and CBZ emerging as particularly interesting candidates for further studies.

Effect of Ultrasound on the Vasculature and Extravasation of Nanoscale Particles Imaged in Real Time.

Ultrasound and microbubbles have been found to improve the delivery of drugs and nanoparticles to tumor tissue. To obtain new knowledge on the influence of vascular parameters on extravasation and to elucidate the effect of acoustic pressure on extravasation and penetration of nanoscale particles into the extracellular matrix, real-time intravital multiphoton microscopy was performed during sonication of tumors growing in dorsal window chambers. The impact of vessel diameter, vessel structure and blood flow was characterized. Fluorescein isothiocyanate-dextran (2 MDa) was injected to visualize blood vessels. Mechanical indexes (MI) of 0.2-0.8 and in-house-made, nanoparticle-stabilized microbubbles or Sonovue were applied. The rate and extent of penetration into the extracellular matrix increased with increasing MI. However, to achieve extravasation, smaller vessels required MIs (0.8) higher than those of blood vessels with larger diameters. Ultrasound changed the blood flow rate and direction. Interestingly, the majority of extravasations occurred at vessel branching points.

Therapeutic Effect of Cabazitaxel and Blood-Brain Barrier opening in a Patient-Derived Glioblastoma Model.

Treatment of glioblastoma and other diseases in the brain is especially challenging due to the blood-brain barrier, which effectively protects the brain parenchyma. In this study we show for the first time that cabazitaxel, a semi-synthetic derivative of docetaxel can cross the blood-brain barrier and give a significant therapeutic effect in a patient-derived orthotopic model of glioblastoma. We show that the drug crosses the blood-brain barrier more effectively in the tumor than in the healthy brain due to reduced expression of p-glycoprotein efflux pumps in the vasculature of the tumor. Surprisingly, neither ultrasound-mediated blood-brain barrier opening (sonopermeation) nor drug formulation in polymeric nanoparticles could increase either accumulation of the drug in the brain or therapeutic effect. This indicates that for hydrophobic drugs, sonopermeation of the blood brain barrier might not be sufficient to achieve improved drug delivery. Nonetheless, our study shows that cabazitaxel is a promising drug for the treatment of brain tumors.

Small variations in nanoparticle structure dictate differential cellular stress responses and mode of cell death.

For optimal exploitation of nanoparticles (NPs) in biomedicine, and to predict nanotoxicity, detailed knowledge of the cellular responses to cell-bound or internalized NPs is imperative. The final outcome of NP-cell interaction is dictated by the type and magnitude of the NP insult and the cellular response. Here, this has been systematically studied by using poly(alkylcyanoacrylate) (PACA) particles differing only in their alkyl side chains; butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), respectively. Surprisingly, these highly similar NPs induced different stress responses and modes of cell death in human cell lines. The POCA particles generally induced endoplasmic reticulum stress and apoptosis. In contrast, PBCA and PEBCA particles induced oxidative stress and lipid peroxidation depending on the level of the glutathione precursor cystine and transcription of the cystine transporter SLC7A11. The latter was induced as a protective response by the transcription factors ATF4 and Nrf2. PBCA particles strongly activated ATF4 downstream of the eIF2α kinase HRI, whereas PEBCA particles more potently induced Nrf2 antioxidant responses. Intriguingly, PBCA particles activated the cell death mechanism ferroptosis; a promising option for targeting multidrug-resistant cancers. Our findings highlight that even minor differences in NP composition can severely impact the cellular response to NPs. This may have important implications in therapeutic settings.

Cabazitaxel-loaded Poly(2-ethylbutyl cyanoacrylate) nanoparticles improve treatment efficacy in a patient derived breast cancer xenograft.

The effect of poly(2-ethyl-butyl cyanoacrylate) nanoparticles containing the cytotoxic drug cabazitaxel was studied in three breast cancer cell lines and one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice. Nanoparticle-encapsulated cabazitaxel had a much better efficacy than similar concentrations of free drug in the basal-like patient-derived xenograft and resulted in complete remission of 6 out of 8 tumors, whereas free drug gave complete remission only with 2 out of 9 tumors. To investigate the different efficacies obtained with nanoparticle-encapsulated versus free cabazitaxel, mass spectrometry quantification of cabazitaxel was performed in mice plasma and selected tissue samples. Nanoparticle-encapsulated drug had a longer circulation time in blood. There was approximately a three times higher drug concentration in tumor tissue 24 h after injection, and two times higher 96 h after injection of nanoparticles with drug compared to the free drug. The tissue biodistribution obtained after 24 h using mass spectrometry analyses correlates well with biodistribution data obtained using IVIS® Spectrum in vivo imaging of nanoparticles labeled with the fluorescent substance NR668, indicating that these data also are representative for the nanoparticle distribution. Furthermore, immunohistochemistry was used to estimate infiltration of macrophages into the tumor tissue following injection of nanoparticle-encapsulated and free cabazitaxel. The higher infiltration of anti-tumorigenic versus pro-tumorigenic macrophages in tumors treated with the nanoparticles might also contribute to the improved effect obtained with the nanoparticle-encapsulated drug. Tumor infiltration of pro-tumorigenic macrophages was four times lower when using nanoparticles containing cabazitaxel than when using particles without drug, and we speculate that the very good therapeutic efficacy obtained with our cabazitaxel-containing particles may be due to their ability to reduce the level of pro-tumorigenic macrophages in the tumor. In summary, encapsulation of cabazitaxel in poly(2-ethyl-butyl cyanoacrylate) nanoparticles seems promising for treatment of breast cancer.