Co-delivery of Docetaxel and Disulfonate Tetraphenyl Chlorin in One Nanoparticle Produces Strong Synergism between Chemo- and Photodynamic Therapy in Drug-Sensitive and -Resistant Cancer Cells.

Cancer therapies based on the combinations of different drugs and/or treatment modalities are emerging as important strategies for increasing efficacy and cure, decreasing unwanted toxicity, and overcoming drug resistance, provided that optimized drug concentration ratios are delivered into the target tissue. To these purposes, delivery systems such as nanoparticles (NPs) offer the unique opportunity to finely tune the drug loading and the release rate of drug combinations in the target tissues. Here, we propose double-layered polymeric NPs for the delivery of the chemotherapeutic docetaxel (DTX) and the photosensitizer disulfonate tetraphenyl chlorin (TPCS2a) coated with hyaluronic acid (HA), which allows cell targeting via CD44 receptors. The simultaneous delivery of the two drugs aims at killing DTX-sensitive (HeLa-P, MDA-MB-231) and DTX-resistant (HeLa-R) cancer cells by combining chemotherapy and photodynamic therapy (PDT). Using the Chou and Talalay method that analyses drug interactions and calculates combination index (CI) using the median-effect principle, we compared the efficiency of DTX chemotherapy combined with TPCS2a-PDT for drugs delivered in the standard solvents, coloaded in the same NP (DTX/TPCS2a-NP) or loaded in separate NPs (DTX-NPs + TPCS2a-NPs). Along with the drug interaction studies, we gained insight into cell death mechanisms after combo-therapy and into the extent of TPCS2a intracellular uptake and localization. In all cell lines considered, the analysis of the viability data revealed synergistic drug/treatment interaction especially when DTX and TPCS2a were delivered to cells coloaded in the same NPs despite the reduced PS uptake measured in the presence of the delivery systems. In fact, while the combinations of the free drugs or drugs in separate NPs gave slight synergism (CI < 1) only at doses killing more than 50% of the cells, the combination of drugs in one NPs gave high synergism also at doses killing 10-20% of the cells. Furthermore, the DTX dose in the combination DTX/TPCS2a-NPs could be reduced by ∼2.6- and 10.7-fold in HeLa-P and MDA-MB-231, respectively. Importantly, drug codelivery in NPs was very efficient in inducing cell mortality also in DTX resistant HeLa-R cells overexpressing P-glycoprotein 1 in which the dose of the chemotherapeutic can be reduced by more than 100 times using DTX/TPCS2a-NPs. Overall, our data demonstrate that the protocol for the preparation of HA-targeted double layer polymeric NPs allows to control the concentration ratio of coloaded drugs and the delivery of the transported drugs for obtaining a highly synergistic interaction combining DTX-chemotherapy and TPCS2a-PDT.

Conjugation of photosensitisers to antimicrobial peptides increases the efficiency of photodynamic therapy in cancer cells.

Some antimicrobial peptides (AMPs) have the ability to penetrate and kill not only pathogenic microorganisms but also cancer cells, while they are less active toward normal eukaryotic cells. Here we have investigated the potential of three AMPs, namely apidaecin 1b (Api), magainin 2 (Mag) and buforin II (Buf), as carriers of drugs for cancer cells by using the hydrophobic photosensitiser 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (cTPP) as the drug model, conjugated to the N-terminus of the peptides. Flow cytometry measurements demonstrated that conjugation of cTPP increased its rate and efficiency of uptake in A549 human lung adenocarcinoma cells in the order Mag > Buf > Api. In vitro photodynamic therapy (PDT) experiments showed that the increased uptake of the conjugated cTPP determined 100% cell killing at concentrations in the nanomolar range while micromolar concentrations were required for the same killing effect with unconjugated cTPP. Serum proteins interacted with cTPP conjugated to Buf and Api and slightly interfered with the cellular uptake of these conjugates but not with that of Mag. The data suggest electrostatic interactions of the conjugates with sialic acid and ganglioside rich domains, as lipid rafts of the plasma membrane, followed by cell internalization via non-caveolar dynamin-dependent endocytosis as indicated by the effects of inhibitors of specific endocytic pathways. Our study demonstrated that the three AMPs investigated, Mag in particular, have the ability to carry a hydrophobic cargo inside cancer cells and may therefore represent useful carriers of anticancer drugs, especially those with a poor capacity to penetrate inside the target cells.

PEGylation of ORMOSIL nanoparticles differently modulates the in vitro toxicity toward human lung cells.

ORganically MOdified SILica (ORMOSIL) nanoparticles (NPs) appear promising carriers for the delivery of drugs to target tissues but concerns on possible cytotoxic effects exist. Here, we studied the in vitro responses to ORMOSIL NPs in different types of human lung cells to determine the effects of polyethylene glycol (PEG) coating on NP cytotoxicity. Non-PEG NPs caused a concentration-dependent decrease of viability of all types of cells, while PEG NPs induced deleterious effects and death in carcinoma alveolar type II A549 cells but not in CCD-34Lu fibroblasts and NCI-H2347 adenocarcinoma cells. Reactive oxygen species were detected in cells incubated with PEG NPs, but their deactivation by superoxide dismutase and catalase did not protect A549 cells from death, suggesting that the oxidative stress was not the main determinant of cytotoxicity. Only in A549 cells PEG NPs modulated the transcription of genes involved in inflammation, signal transduction and cell death. Transmission electron microscopy evidenced a unique intracellular localization of PEG NPs in the lamellar bodies of A549 cells, which could be the most relevant factor leading to cytotoxicity by reducing the production of surfactant proteins and by interfering with the pulmonary surfactant system.

A Comparative Study on Two Cationic Porphycenes: Photophysical and Antimicrobial Photoinactivation Evaluation.

Over the last decades, the number of pathogenic multi-resistant microorganisms has grown dramatically, which has stimulated the search for novel strategies to combat antimicrobial resistance. Antimicrobial photodynamic therapy (aPDT) is one of the promising alternatives to conventional treatments based on antibiotics. Here, we present a comparative study of two aryl tricationic porphycenes where photoinactivation efficiency against model pathogenic microorganisms is correlated to the photophysical behavior of the porphycene derivatives. Moreover, the extent of photosensitizer cell binding to bacteria has been assessed by flow cytometry in experiments with, or without, removing the unbound porphycene from the incubation medium. Results show that the peripheral substituent change do not significantly affect the overall behavior for both tricationic compounds neither in terms of photokilling efficiency, nor in terms of binding.

Folate-targeted PEGylated liposomes improve the selectivity of PDT with meta-tetra(hydroxyphenyl)chlorin (m-THPC).

The folate receptor (FR) is over-expressed in many human tumours and is being intensively studied also in the field of nanomedicine as a target to enhance the selectivity of drug delivery to cancer cells by using nanocarriers bearing folic acid (FA) on their surface. In this study we report the encapsulation of the photosensitizer (PS) meta-tetra(hydroxyphenyl)chlorin (m-THPC) in FA-targeted PEGylated liposomes used as a novel drug delivery system for photodynamic therapy (PDT) of cancer. Our in vitro investigations revealed that only a modest fraction of targeted liposomes were internalized by specific endocytosis in FR-positive KB cells. However, FA-liposomes doubled the uptake of the entrapped m-THPC with respect to un-targeted liposomes and enhanced the photo-induced cytotoxicity in KB cells. In contrast, in FR-negative A549 cells FA-targeted or un-targeted liposomes exhibited a very similar extent of internalization and as a consequence the same photo-killing efficiency.

The toxicity outcome of silica nanoparticles (Ludox®) is influenced by testing techniques and treatment modalities.

We analyzed the influence of the kind of cytotoxicity test and its application modality in defining the level of hazard of the in vitro exposures to nanostructures. We assessed the cytotoxicity induced by two different Ludox® silica nanoparticles (NPs), AS30 and SM30, on three human cell lines, CCD-34Lu, A549, and HT-1080. Dynamic light scattering measurements showed particle agglomeration when NPs are diluted in culture medium supplemented with fetal calf serum. We examined the impact of such particle aggregation on the cytotoxicity by exposing the cells to NPs under different treatment modalities: short incubation (2 h) in serum-free medium or long incubation (24-72 h) in serum-containing medium. Under this last modality, NP suspensions tended to form aggregates and were toxic at concentrations five- to tenfold higher than in serum-free medium. The results of cell survival varied considerably when the long-term clonogenic assay was performed to validate the data of the short-term MTS assay. Indeed, the half maximum effective concentrations (EC(50)) in all the three cell lines were four- to fivefold lower when calculated from the data of clonogenic assay than of MTS. Moreover, the mechanisms of NP toxicity were cell-type-specific, showing that CCD-34Lu are prone to the induction of plasma membrane damages and HT-1080 are prone to DNA double-strand break and apoptosis induction. Taken together, our results demonstrate that the choice of testing strategy and treatment conditions plays an important role in assessing the in vitro toxicity of NPs.

Overview of Nanoparticle-Based Approaches for the Combination of Photodynamic Therapy (PDT) and Chemotherapy at the Preclinical Stage.

The widespread diffusion of photodynamic therapy (PDT) as a clinical treatment for solid tumors is mainly limited by the patient's adverse reaction (skin photosensivity), insufficient light penetration in deeply seated neoplastic lesions, unfavorable photosensitizers (PSs) biodistribution, and photokilling efficiency due to PS aggregation in biological environments. Despite this, recent preclinical studies reported on successful combinatorial regimes of PSs with chemotherapeutics obtained through the drugs encapsulation in multifunctional nanometric delivery systems. The aim of the present review deals with the punctual description of several nanosystems designed not only with the objective of co-transporting a PS and a chemodrug for combination therapy, but also with the goal of improving the therapeutic efficacy by facing the main critical issues of both therapies (side effects, scarce tumor oxygenation and light penetration, premature drug clearance, unspecific biodistribution, etc.). Therefore, particular attention is paid to the description of bio-responsive drugs and nanoparticles (NPs), targeted nanosystems, biomimetic approaches, and upconverting NPs, including analyzing the therapeutic efficacy of the proposed photo-chemotherapeutic regimens in in vitro and in vivo cancer models.

Biodegradable nanoparticles combining cancer cell targeting and anti-angiogenic activity for synergistic chemotherapy in epithelial cancer.

A biodegradable engineered nanoplatform combining anti-angiogenic activity and targeting of cancer cells to improve the anticancer activity of docetaxel (DTX) is here proposed. Indeed, we have developed biodegradable nanoparticles (NPs) of poly(ethylene glycol)-poly(ε-caprolactone), exposing on the surface both folate motifs (Fol) for recognition in cells overexpressing Folate receptor-α (FRα) and the anti-angiogenic hexapeptide aFLT1. NPs showed a size around 100 nm, the exposure of 60% of Fol moieties on the surface, and the ability to entrap DTX and sustain its release with time. NPs were stable in simulated biological fluids and slightly interacted with Fetal Bovine serum, especially in the formulation decorated with Fol and aFLT1. The presence of Fol on NPs did not impair the anti-angiogenic activity of aFLT1, as assessed by in vitro tube formation assay in HUVEC endothelial cells. In both 2D and 3D KB cell cultures in vitro, the cytotoxicity of DTX loaded in NPs was not significantly affected by Fol/aFLT1 double decoration compared to free DTX. Remarkably, NPs distributed differently in 3D multicellular spheroids of FRα-positive KB cancer cells depending on the type of ligand displayed on the surface. In particular, NPs unmodified on the surface were randomly distributed in the spheroid, whereas the presence of Fol promoted the accumulation in the outer rims of the spheroid. Finally, NPs with Fol and aFLT1 gave a uniform distribution throughout the spheroid structure. When tested in zebrafish embryos xenografted with KB cells, NPs displaying Fol/aFLT1 reduced DTX systemic toxicity and inhibited the growth of the tumor mass and associated vasculature synergistically. Overall, nanotechnology offers excellent ground for combining therapeutic concepts in cancer, paving the way to novel multifunctional nanopharmaceuticals decorated with bioactive elements that can significantly improve therapeutic outcomes.

Doxorubicin-NO Releaser Molecular Hybrid Activatable by Green Light to Overcome Resistance in Breast Cancer Cells.

The biological activity of a molecular hybrid (DXNO-GR) joining doxorubicin (DOX) and an N-nitroso moiety releasing nitric oxide (NO) under irradiation with the biocompatible green light has been investigated against DOX-sensitive (MCF7) and -resistant (MDA-MB-231) breast cancer cells in vitro. DXNO-GR shows significantly higher cellular internalization than DOX in both cell lines and, in contrast to DOX, does not experience cell efflux in MDR overexpressing MDA-MB-231 cells. The higher cellular internalization of the DXNO-GR hybrid seems to be mediated by bovine serum albumin (BSA) as a suitable carrier among serum proteins, according to the high binding constant measured for DXNO-GR, which is more than one order of magnitude larger than that reported for DOX. Despite the higher cellular accumulation, DXNO-GR is not toxic in the dark but induces remarkable cell death following photoactivation with green light. This lack of dark toxicity is strictly related to the different cellular compartmentalization of the molecular hybrid that, different from DOX, does not localize in the nucleus but is mainly confined in the Golgi apparatus and endoplasmic reticulum and therefore does not act as a DNA intercalator. The photochemical properties of the hybrid are not affected by binding to BSA as demonstrated by the direct detection of NO photorelease, suggesting that the reduction of cell viability observed under light irradiation is a combined effect of DOX phototoxicity and NO release which, ultimately, inhibits MDR1 efflux pump in DOX-resistant cells.

Meta-tetra(hydroxyphenyl)chlorin-loaded liposomes sterically stabilised with poly(ethylene glycol) of different length and density: characterisation, in vitro cellular uptake and phototoxicity.

We studied the effects of density and thickness of PEG coating on in vitro cellular uptake, and dark- and photo-toxicity of liposomal formulations (Fospeg) of the photodynamic agent meta-tetrahydroxyphenyl chlorin (m-THPC). The cellular uptake of various Fospeg formulations was determined by flow cytometry in CCD-34Lu human normal fibroblasts and A549 lung cancer cells. Dark and light-induced cytotoxicity was measured by MTS assay after exposure to increasing concentrations of Fospeg only and followed by irradiation with red light. Intracellular localization of m-THPC delivered by Fospeg was determined by fluorescence microscopy. The studies were carried out in comparison with m-THPC delivered by the standard solvent. In the dark all Fospeg formulations were less cytotoxic than m-THPC in standard solvent (ethanol/poly(ethylene glycol 400/water; 20 : 30 : 50 by vol.) and cytotoxicity decreased by increasing PEGylation. m-THPC delivered as Fospeg was internalised by endocytosis and localised mainly in the Golgi apparatus and endoplasmic reticulum. The efficiency of cellular uptake of Fospeg was reduced by 30-40% with respect to m-THPC in standard solution causing a slight reduction of the phototoxicity but without serious impairment of the efficacy of the treatment. Our study suggests that PEGylated liposomes are promising nanocarriers for the delivery of photosensitisers for photodynamic therapy because they reduce dark cytotoxicity while preserving therapeutic efficacy.

A generator of peroxynitrite activatable with red light.

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) as "unconventional" therapeutics with precise spatiotemporal control by using light stimuli may open entirely new horizons for innovative therapeutic modalities. Among ROS and RNS, peroxynitrite (ONOO-) plays a dominant role in chemistry and biology in view of its potent oxidizing power and cytotoxic action. We have designed and synthesized a molecular hybrid based on benzophenothiazine as a red light-harvesting antenna joined to an N-nitroso appendage through a flexible spacer. Single photon red light excitation of this molecular construct triggers the release of nitric oxide (˙NO) and simultaneously produces superoxide anions (O2˙-). The diffusion-controlled reaction between these two radical species generates ONOO-, as confirmed by the use of fluorescein-boronate as a highly selective chemical probe. Besides, the red fluorescence of the hybrid allows its tracking in different types of cancer cells where it is well-tolerated in the dark but induces remarkable cell mortality under irradiation with red light in a very low concentration range, with very low light doses (ca. 1 J cm-2). This ONOO- generator activatable by highly biocompatible and tissue penetrating single photon red light can open up intriguing prospects in biomedical research, where precise and spatiotemporally controlled concentrations of ONOO- are required.

Keratin nanoparticles and photodynamic therapy enhance the anticancer stem cells activity of salinomycin.

The high rates of aggressiveness, drug resistance and relapse of breast cancer (BC) are mainly attributed to the inability of conventional therapies to equally eradicate bulk differentiated cells and cancer stem cells (CSCs). To improve the effectiveness of BC treatments, we report the in-water synthesis of novel keratin-based nanoformulations, loaded with the CSC-specific drug salinomycin (SAL), the photosensitizer chlorin e6 (Ce6) and vitamin E acetate (SAL/Ce6@kVEs), which combine the capability of releasing SAL with the production of singlet oxygen upon light irradiation. In vitro experiments on BC cell lines and CSC-enriched mammospheres exposed to single or combined therapies showed that SAL/Ce6@kVEs determine synergistic cell killing, limit their self-renewal capacity and decrease the stemness potential by eradication of CSCs. In vivo experiments on zebrafish embryos confirmed the capacity of SAL nanoformulations to interfere with the Wnt/ß-catenin signaling pathway, which is dysregulated in BC, thus identifying a target for further translation into pre-clinical models.

CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro.

The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a therapeutic strategy based on the simultaneous delivery of docetaxel (DTX) and the photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a) using hyaluronic acid (HA) coated polymeric nanoparticles (HA-NPs) for the targeting and killing of CD44 over-expressing breast cancer (BC) cells, both differentiated and CSCs (CD44high/CD24low population), thus combining chemotherapy and photodynamic therapy (PDT). Using the CD44high MDA-MB-231 and the CD44low MCF-7 cells, we demonstrated the occurrence of CD44-mediated uptake of HA-NPs both in monolayers and mammosphere cultures enriched in CSCs. Cell treatments showed that combination therapy using co-loaded NPs (HA@DTX/TPCS2a-NPs) had superior efficacy over monotherapies (HA@DTX-NPs or HA@TPCS2a-NPs) in reducing the self-renewal capacity, measured as mammosphere formation efficiency, and in eradicating the CSC population evaluated with aldehyde dehydrogenase activity assay and CD44/CD24 immunostaining. In summary, these in vitro studies demonstrated for the first time the potential of the combination of DTX-chemotherapy and TPCS2a-PDT for killing CSCs using properly designed NPs.

The cellular uptake of meta-tetra(hydroxyphenyl)chlorin entrapped in organically modified silica nanoparticles is mediated by serum proteins.

Nanosized objects made of various materials are gaining increasing attention as promising vehicles for the delivery of therapeutic and diagnostic agents for cancer. Photodynamic therapy (PDT) appears to offer a very attractive opportunity to implement drug delivery systems since no release of the sensitizer is needed to obtain the therapeutic effect and the design of the nanovehicle should be much easier. The aim of our study was to investigate the use of organic-modified silica nanoparticles (NPs) for the delivery of the second-generation photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC) to cancer cells in vitro. mTHPC was entrapped in NPs (approximately 33 nm diameter) in a monomeric form which produced singlet oxygen with a high efficiency. In aqueous media with high salt concentrations, the NPs underwent aggregation and precipitation but their stability could be preserved in the presence of foetal bovine serum. The cellular uptake, localization and phototoxic activity of mTHPC was determined comparatively in human oesophageal cancer cells after its delivery by the NPs and the standard solvent ethanol/poly(ethylene glycol) 400/water (20:30:50, by vol). The NP formulation reduced the cellular uptake of mTHPC by about 50% in comparison to standard solvent while it did not affect the concentration-dependent photokilling activity of mTHPC and its intracellular localization. Fluorescence resonance energy transfer measurements, using NPs with mTHPC physically entrapped and a cyanine covalently linked, and ultracentrifugation experiments indicated that mTHPC is transferred from NPs to serum proteins when present in the medium. However, the coating of the NP surface with poly(ethylene glycol) largely prevented the transfer to proteins. In conclusion, mTHPC is rapidly transferred from the uncoated nanoparticles to the serum proteins and then internalized by the cells as a protein complex, irrespective of its modality of delivery.

Keratin nanoparticles co-delivering Docetaxel and Chlorin e6 promote synergic interaction between chemo- and photo-dynamic therapies.

The combination of chemotherapy and photodynamic therapy (PDT) is considered a valuable strategy for increasing therapeutic response in cancer treatment, and the re-formulation of pharmaceuticals in biocompatible nanoparticles (NPs) is particularly appealing for the possibility of co-loading drugs exerting cytotoxicity by different mechanisms, with the aim to produce synergic effects. We report the in-water synthesis of a novel keratin-based nanoformulation for the co-delivery of the antimitotic Docetaxel (DTX) and the photosensitizer Chlorin e6 (Ce6). The drug-induced aggregation method allowed the formation of monodisperse NPs (DTX/Ce6-KNPs) with an average diameter of 133 nm and loaded with a drug ratio of 1:1.8 of Ce6 vs DTX. The efficacy of DTX/Ce6-KNPs was investigated in vitro in monolayers and spheroids of DTX-sensitive HeLa (HeLa-P) and DTX-resistant HeLa (HeLa-R) cells. In monolayers, the cytotoxic effects of DTX/Ce6-KNPs toward HeLa-P cells were comparable to those induced by free DTX + Ce6, while in HeLa-R cells the drug co-loading in KNPs produced synergic interaction between chemotherapy and PDT. Moreover, as respect to monotherapies, DTX/Ce6-KNPs induced stronger cytotoxicity to both HeLa-P and HeLa-R multicellular spheroids and reduced their volumes up to 50%. Overall, the results suggest that KNPs are very promising systems for the co-delivery of chemotherapeutics and PSs, favoring synergic interactions between PDT and chemotherapy.

Biodegradable nanoparticles exposing a short anti-FLT1 peptide as antiangiogenic platform to complement docetaxel anticancer activity.

Inhibition of tumor angiogenesis is considered as a valuable clinical strategy to treat some tumors, although benefits in term of progression-free and overall survival have been modest. Recent findings have pushed toward the use of antiangiogenic drugs in combination with chemotherapy regimens to potentiate therapeutic outcome. Herein, we propose a novel type of biodegradable antiangiogenic core-shell polymeric nanoparticles (NPs) for the delivery of poorly water-soluble chemotherapeutics. An amphiphilic diblock copolymer of poly(ethyleneglycol)-poly(ε-caprolactone) (PEG-PCL) was conjugated with an anti-FLT1 hexapeptide (aFLT1) at -OH PEG end, mixed in appropriate ratios with a monomethoxy-PEG-PCL and nanoprecipitated to form core-shell aFLT1-bearing NPs (DBLaFLT1). DBLaFLT1 were <100 nm, exposed aFLT1 on the surface and showed a higher thickness of the external hydrophilic shell as compared to NPs that do not bear aFLT1 (DBL). Very interestingly, DBLaFLT1 showed an antiangiogenic activity in the human umbilical endothelial cells (HUVEC) tube formation assay three-fold higher than an equivalent dose of free aFLT1. To provide a proof-of-concept of DBLaFLT1 potential in the delivery of conventional chemotherapeutics, docetaxel (DTX) was selected as model drug. DBLaFLT1 entrapped DTX with high efficiency and sustained its release along time in simulated biological conditions. At a non-cytotoxic dose, DTX-loaded DBLaFLT1 almost completely abolished tube formation in HUVEC while inhibition of DTX loaded DBL was significantly lower. The cytotoxicity of DTX-loaded NPs in HUVEC and triple negative breast cancer cells (MDA-MB-231) was not significantly different from that of the free drug in a wide range of concentrations and up to 72 h. Studies carried out in MDA-MB-231 cells implanted in chicken embryo chorioallantoic membranes (CAMs) evidenced an antiangiogenic activity of DTX-loaded DBLaFLT1 higher as compared with that of both DTX-loaded DBL and free DTX. While cancer cell migration from the tumor site was unaffected, the anticancer activity of DTX-loaded NPs was higher than that of free DTX and maximized for DTX-DBLaFLT1. In perspective, these results suggest that the delivery approach proposed here can be applied to other lipophilic chemotherapeutics devoid of relevant antiangiogenic properties to improve the final therapeutic response.

Shedding light on surface exposition of poly(ethylene glycol) and folate targeting units on nanoparticles of poly(ε-caprolactone) diblock copolymers: Beyond a paradigm.

Polymeric nanoparticles (NPs) of poly(ε-caprolactone) (PCL) covered with a hydrophilic poly(ethylene glycol) (PEG) shell are usually prepared from diblock PEG-PCL copolymers through different techniques. Furthermore PEG, NPs can be decorated with targeting ligands to accumulate in specific cell lines. However, the density and conformation of PEG on the surface and its impact on the exposition of small targeting ligands has been poorly considered so far although this has a huge impact on biological behaviour. Here, we focus on PEG-PCL NPs and their folate-targeted version to encourage accumulation in cancer cells overexpressing folate receptor α. NPs were prepared with mixtures of PEG-PCL with different PEG length (short 1.0kDa, long 2.0kDa,) and a folate-functionalized PEG-PCL (PEG 1.5kDa) by the widely employed solvent displacement method. In depth characterization of NPs surface by 1H NMR, fluorescence and photon correlation spectroscopy evidenced a PEGylation extent below 7% with PEG in a mushroom conformation and the presence of folate more exposed to water pool in the case of copolymer with short PEG. NPs with short PEG adsorbed HSA forming a soft corona without aggregating. Although limited, PEGylation overall reduced NPs uptake in human macrophages. Uptake of NPs exposing folate prepared with short PEG was higher in KB cells (FR+) than in A549 (FR-), occurred via FR-receptor and involved lipid rafts-dependent endocytosis. In conclusion, the present results demonstrate that PEG length critically affects protein interaction and folate exposition with a logical impact on receptor-mediated cell uptake. Our study highlights that the too simplistic view suggesting that PEG-PCL gives PEG-coated NPs needs to be re-examined in the light of actual surface properties, which should always be considered case-by-case.

Low doses of cisplatin or gemcitabine plus Photofrin/photodynamic therapy: Disjointed cell cycle phase-related activity accounts for synergistic outcome in metastatic non-small cell lung cancer cells (H1299).

We compared the effects of monotherapy (photodynamic therapy or chemotherapy) versus combination therapy (photodynamic therapy plus a specific drug) on the non-small cell lung cancer cell line H1299. Our aim was to evaluate whether the additive/synergistic effects of combination treatment were such that the cytostatic dose could be reduced without affecting treatment efficacy. Photodynamic therapy was done by irradiating Photofrin-preloaded H1299 p53/p16-null cells with a halogen lamp equipped with a bandpass filter. The cytotoxic drugs used were cis-diammine-dichloroplatinum [II] (CDDP or cisplatin) and 2',2'-difluoro-2'-deoxycytidine (gemcitabine). Various treatment combinations yielded therapeutic effects (trypan blue dye exclusion test) ranging from additive to clearly synergistic, the most effective being a combination of photodynamic therapy and CDDP. To gain insight into the cellular response mechanisms underlying favorable outcomes, we analyzed the H1299 cell cycle profiles and the expression patterns of several key proteins after monotherapy. In our conditions, we found that photodynamic therapy with Photofrin targeted G0-G1 cells, thereby causing cells to accumulate in S phase. In contrast, low-dose CDDP killed cells in S phase, thereby causing an accumulation of G0-G1 cells (and increased p21 expression). Like photodynamic therapy, low-dose gemcitabine targeted G0-G1 cells, which caused a massive accumulation of cells in S phase (and increased cyclin A expression). Although we observed therapeutic reinforcement with both drugs and photodynamic therapy, reinforcement was more pronounced when the drug (CDDP) and photodynamic therapy exert disjointed phase-related cytotoxic activity. Thus, if photodynamic therapy is appropriately tuned, the dose of the cytostatic drug can be reduced without compromising the therapeutic response.

Uptake and photo-toxicity of Foscan®, Foslip® and Fospeg® in multicellular tumor spheroids.

In cancer photodynamic therapy (PDT), an efficient and homogeneous intratumoral accumulation of the photosensitizer (PS) is required to induce cell damages in the entire tumor mass after light activation. Thus, in this study we investigated penetration ability and photodynamic efficiency of meta-tetra(hydroxyphenyl)chlorin (m-THPC) in standard formulation (Foscan®) and in its non PEGylated and PEGylated liposomal formulations, Foslip® and Fospeg®, in HeLa multicellular spheroids, as in vitro avascular models of solid tumors. Confocal microscopy studies demonstrated that m-THPC fluorescence was confined in the external cell layers of spheroids with a slightly higher accumulation of Foslip® and Fospeg® with respect to Foscan®. Irradiation with red light, following 24h incubation of spheroids with the m-THPC formulations, caused however photodamages also in cells located in the central part of spheroids, as documented by transmission electron microscopy analyses. Overall, the photodynamic effects of the three m-THPC formulations on HeLa cell spheroids were comparable in terms of cell viability measured with the MTS assay. It is however worth noting that the delivery of m-THPC by liposomes significantly abolished its cytotoxicity in the dark, slightly improved the cellular uptake and, following PDT, promoted cell loss and spheroid disassembling to a higher extent when compared to Foscan®.

Pluronic® P123/F127 mixed micelles delivering sorafenib and its combination with verteporfin in cancer cells.

Here, we developed Pluronic® P123/F127 (poloxamer) mixed micelles for the intravenous delivery of the anticancer drug sorafenib (SRB) or its combination with verteporfin (VP), a photosensitizer for photodynamic therapy that should complement well the cytotoxicity profile of the chemotherapeutic. SRB loading inside the core of micelles was governed by the drug:poloxamer weight ratio, while in the case of the SRB-VP combination, a mutual interference between the two drugs occurred and only specific ratios could ensure maximum loading efficiency. Coentrapment of SRB did not alter the photophysical properties of VP, confirming that SRB did not participate in any bimolecular process with the photosensitizer. Fluorescence resonance energy-transfer measurement of micelles in serum protein-containing cell-culture medium demonstrated the excellent stability of the system in physiologically relevant conditions. These results were in line with the results of the release study showing a release rate of both drugs in the presence of proteins slower than in phosphate buffer. SRB release was sustained, while VP remained substantially entrapped in the micelle core. Cytotoxicity studies in MDA-MB231 cells revealed that at 24 hours, SRB-loaded micelles were more active than free SRB only at very low SRB concentrations, while at 24+24 hours a prolonged cytotoxic effect of SRB-loaded micelles was observed, very likely mediated by the block in the S phase of the cell cycle. The combination of SRB with VP under light exposure was less cytotoxic than both the free combination and VP-loaded micelles + SRB-loaded micelles combination. This behavior was clearly explainable in terms of micelle uptake and intracellular localization. Besides the clear advantage of delivering SRB in poloxamer micelles, our results provide a clear example that each photochemotherapeutic combination needs detailed investigations on their particular interaction, and no generalization on enhanced cytotoxic effects should be derived a priori.