Rimona Margalit

Nanocarriers as an emerging platform for cancer therapy

Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients. Several therapeutic nanocarriers have been approved for clinical use. However, to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells. This review examines some of the approved formulations and discusses the challenges in translating basic research to the clinic. We detail the arsenal of nanocarriers and molecules available for selective tumour targeting, and emphasize the challenges in cancer treatment.

Loading mitomycin C inside long circulating hyaluronan targeted nano-liposomes increases its antitumor activity in three mice tumor models.

The frequent overexpression of the hyaluronan receptors CD44 and RHAMM in cancer cells opens the door for targeting by the naturally‐occurring high‐Mr hyaluronan. This is the first time effective in vivo tumor targeting is reported for mitomycin C (MMC) loaded inside nano‐sized hyaluronan‐liposomes (denoted tHA‐LIP). The severe adverse effects of free MMC made it a rational candidate for an effective targeted carrier. In vitro, loading MMC inside tHA‐LIP increased drug potency 100‐fold, in cells overexpressing, but not in cells underexpressing, hyaluronan receptors. Both types of liposomes were non‐toxic and reduced MMC‐related toxicity in healthy C57BL/6 mice. In 3 tumor models, BALB/c bearing C‐26 solid tumors; C57BL/6 bearing B16F10.9 or (separately) D122 lung metastasis, tHA‐LIP were long‐circulating, 7‐fold and 70‐fold longer than nt‐LIP and free MMC, respectively. tHA‐LIP‐mediated MMC accumulation in tumor‐bearing lungs was 20% of injected dose, compared to 0.6% and 4% with free drug and nt‐LIP, respectively. Tumor‐free lungs showed low accumulation, irrespective of drug formulation. Key indicators of therapeutic responses, tumor progression, metastatic burden and survival, were superior (p < 0.001) in animals receiving MMC‐loaded tHA‐LIP, no treatment, MMC‐loaded nt‐LIP and free drug. In conclusion, tHA‐LIP perform as tumor‐targeted carriers, with promising prospects for treatment of tumors overexpressing hyaluronan receptors.

Hyaluronan is a key component in cryoprotection and formulation of targeted unilamellar liposomes

Lyophilized unilamellar liposomes (ULV), the dosage form of choice for shelf-life, revert upon reconstitution to the larger multilamellar liposomes (MLV), which is detrimental to the many carrier-mediated therapies that require small particles. High doses of sugars such as trehalose, sucrose and others, included in the original formulations for cryoprotection, were shown to prevent the conversion to MLV. In this study we set out to test whether hyaluronan (HA), the surface-bound ligand in our previously developed targeted bioadhesive liposomes (BAL), can also act as a cryoprotectant. The studies included structural and physicochemical characterization of original and reconstituted hyaluronan-ULV (HA-ULV). For each HA-ULV, similar regular ULV (RL-ULV) served as controls. Four properties were tested: particle size, zeta potential, encapsulation efficiency and half-life of drug release (tau(1/2)), for three drugs-chloramphenicol (CAM), vinblastine (VIN) and mitomycin C (MMC). Encapsulation efficiencies of the original systems were quite alike for similar RL-ULV and HA-ULV ranging from 25% to 70%. All systems acted as sustained-release drug depots, tau(1/2) ranging from 1.3 to 5.3 days. Drug species and lipid composition were the major determinants of encapsulation and release magnitudes. By all tests, as anticipated, lyophilization generated significant changes in the reconstituted RL-ULV: 17-fold increase in diameter; tripling of zeta potential; 25-60% drop in encapsulation efficiencies; 25-30% decrease in tau(1/2). In contrast, the reconstituted HA-ULV retained the same dimensions, zeta potentials, encapsulation efficiencies and tau(1/2) of the original systems. These data clearly show HA to be a cryoprotectant, adding another clinically relevant advantage to HA-BAL. We propose that, like the sugars, HA cryoprotects by providing substitute structure-stabilizing H-bonds.

Fluoxetine inhibits multidrug resistance extrusion pumps and enhances responses to chemotherapy in syngeneic and in human xenograft mouse tumor models

Multidrug resistance (MDR) operated by extrusion pumps such as P-glycoprotein and multidrug-resistance-associated-proteins, is a major reason for poor responses and failures in cancer chemotherapy. MDR modulators (chemosensitizers) were found among drugs approved for noncancer indications and their derivatives. Yet toxicity, adverse effects, and poor solubility at doses required for MDR reversal prevent their clinical application. Among newly designed chemosensitizers, some still suffer from toxicity and adverse effects, whereas others progressed to clinical trials. Diversities among tumors and among MDR pumps indicate a need for several clinically approved MDR modulators. Here we report for the first time that fluoxetine (Prozac), the well-known antidepressant, is a highly effective chemosensitizer. In vitro, fluoxetine enhanced (10- to 100-fold) cytotoxicity of anticancer drugs (doxorubicin, mitomycin C, vinblastine, and paclitaxel) in drug-resistant but not in drug-sensitive cells (5 and 3 lines, respectively). Fluoxetine increased drug accumulation within MDR-cells and inhibited drug efflux from those cells. In vivo, fluoxetine enhanced doxorubicin accumulation within tumors (12-fold) with unaltered pharmacokinetics. In four resistant mouse tumor models of both syngeneic and human xenograft, combination treatment of fluoxetine and doxorubicin generated substantial (P < 0.001) improvements in tumor responses and in survivals (2- to 3-fold). Moreover, fluoxetine reversed MDR at doses that are well below its human safety limits, free of the severe dose-related toxicity, adverse effects, and poor solubility that are obstacles to other chemosensitizers. This low-dose range, together with the findings reported here, indicate that fluoxetine has a high potential to join the arsenal of MDR reversal agents that may reach the clinic.

Thermodynamics of the redox reaction of cytochromes c of five different species

The redox potential of cytochrome c has interested many investigators, because of its possible significance in understanding both the biological role played by this molecule, and also the relationship between its structure and physicochemical properties [l] . A better insight into this relationship can be gained through knowledge of enthalpy and entropy changes of the redox reaction. Such data were not available until recent calorimetric determinations by George et al. [2] The evaluation of the heat change for several cytochromes c, made by applying the Van? Hoff isochore to equilibrium measurements at various temperatures, is reported in this paper.

Liposomal drug delivery : thermodynamic and chemical kinetic considerations

Abstract The thermodynamics of encapsulation and the kinetics of efflux were studied for a series of small molecular-weight drugs in multilamellar and unilamellar liposomes. Liposome concentration and drug partition coefficient are proposed to be the dominant factors in drug encapsulation. The theoretical basis for this conclusion is presented together with supportive experimental data for progesterone, vinblastine, pilocarpine, serotonin, leu-enkephalin and a model tripeptide. The kinetics and mechanism of drug efflux were evaluated according to Eyrings absolute rate theory. For all systems studied, the data supported a mechanism of two parallel, first-order processes for the efflux of the encapsulated and the unencapsulated drug, respectively. An inverse dependence, which is proposed to be an expression of deviations from ideality, was found between the rate constant for the efflux of the encapsulated drug and liposome concentration. At liposome concentrations of 100 to 150 (mM lipid), the rate constants for efflux of encapsulated progesterone, vinblastine, serotonin and pilocarpine from MLV are 6 × 10−4, 3 × 10−3, 1.3 × 10−2 and 7 × 10−2 (h−1), respectively. Under similar experimental conditions, the corresponding data for unilamellar liposomes are 7 × 10−4, 0.3 and 0.4 (h−1) for progesterone, serotonin and pilocarpine, respectively. The application of kinetic studies of this type for the optimization of shelf-life conditions of liposome/drug systems with respect to drug retention is presented and discussed.

Bioadhesive, collagen-modified liposomes: Molecular and cellular level studies on the kinetics of drug release and on binding to cell monolayers

Liposomes, modified by covalently-anchoring collagen to their surface, were investigated for their abilities to be bioadhesive and to act as sustained-release drug carriers. These bioadhesive liposomes have the potential to induce significant improvements in topical and regional therapies. The major findings for uni-(ULV) and multilamellar (MLV) bioadhesive liposomes are: (a) Both ULV and MLV release small molecular weight drugs over prolonged periods. For example, rate constants of (6 +/- 0.5) x 10(-3) and (2.6 +2- 0.8) x 10(-3) h-1, were obtained for the release of vinblastine and fluconazole, respectively, from collagen-ULV. (b) For a given drug, that rate constant can be shifted (up or down) by the choice of liposome type and collagen-surface density and the latter, if high enough, lead to the formation of an additional liposome-associated drug reservoir. (c) Using monolayers of the A431 cell line to model the in vivo targets, the bioadhesive (but not the regular) liposomes were found to bind with high affinity to the monolayers. For example, equilibrium dissociation constants of 6.3(+/- 3) microM and 2.7(+/- 0.5) microM were determined for bioadhesive MLV and ULV, respectively, with corresponding saturation occupancies of 3.7(+/- 1) and 4.0(+/- 0.2) pmoles liposomal collagen/monolayer of 10(5) cells. (d) Following the retention of bioadhesive MLV at A431 monolayers for 24 h, it was found that: at 4 degrees C, 24 h did not suffice to reach equilibrium, but at 37 degrees C equilibrium binding was obtained within 3-5 h and there was quantitative liposome retention (per viable monolayer) thereafter. It is concluded that these liposomes are bioadhesive sustained-release carriers, as desired, meriting further cellular and in vivo studies.

Studies of hematoporphyrin and hematoporphyrin derivative equilibria in heterogeneous systems. Porphyrin-liposome binding and porphyrin aqueous dimerization

Two processes of porphyrins in heterogeneous systems containing aqueous and membrane phases have been studied with hematoporphyrin and hematoporphyrin derivative: Dimerization equilibrium in the aqueous phases and porphyrin-membrane binding equilibrium using liposomes as models for biological membranes. The interrelationship of aqueous aggregations and membrane binding was probed and the porphyrin aggregation state in the membrane, at equilibrium, was assessed. Fluorimetric techniques were employed. The dimerization equilibrium constants, at neutral pH and 37 degrees C were found to be 2.8 X 10(5) M-1 and 1.9 X 10(6) M-1 for hematoporphyrin and its derivative, respectively. Over a porphyrin concentration range going from monomer-dominant to dimer-dominant systems, we have found that only monomers are bound to the membrane. The respective monomer-liposome binding constants, found to be independent of the initial monomer/dimer distribution in the aqueous phase, were determined to be 1.6 X 10(3) M-1 and 4.1 X 10(3) M-1 at neutral pH and 37 degrees C for hematoporphyrin and its derivative, respectively. The monomer-liposome interaction was found to perurb the initial monomer/dimer distribution in the aqueous phase, so that the monomers residing at equilibrium in the membrane originate from both monomers and dimers in the aqueous phase.

Treatment of resistant human colon cancer xenografts by a fluoxetine–doxorubicin combination enhances therapeutic responses comparable to an aggressive bevacizumab regimen

Pre-clinical studies of multidrug resistance (MDR) usually address severe resistance, yet moderate MDR is already clinically-impeding. The purpose of this study was to characterize moderate drug resistance in human colon cancer, and its modulation by fluoxetine. In vitro fluoxetine enhanced doxorubicins cytotoxicity (10-fold), increased doxorubicins intracellular accumulation (32%) and decreased efflux of intracellular doxorubicin (70%). In vivo, mild treatment with a doxorubicin-fluoxetine combination slowed-down tumor progression significantly (p<0.001 vs. doxorubicin alone), comparable to aggressive treatment with bevacizumab. Collectively, our results suggest that combinations of fluoxetine with chemotherapeutic drugs (P-glycoprotein substrates) are worthy of further pursuit for moderate MDR in the clinic.

THERMODYNAMICS OF PORPHYRIN BINDING TO SERUM ALBUMIN: EFFECTS OF TEMPERATURE, OF PORPHYRIN SPECIES and OF ALBUMIN‐CARRIED FATTY ACIDS

Abstract Porphyrin binding to serum albumin was studied at the molecular level probing the effects of: porphyrin self‐aggregation, porphyrin species, temperature and protein‐bound fatty acids. Human serum albumin was found to have a single high‐affinity site for porphyrin monomers, with binding constants of 2 x 106, 5 x 107 and 3 x 108 (37o C, neutral pH, M−1), for hemato‐, deutero‐ and protoporphyrins, respectively. Three equilibria models for the dimer binding were developed and tested. The data were found to fit best with a model proposing a single high‐affinity binding site for the dimer, independent of and different than the monomer site. The binding constants of the hematoporphyrin and deuteroporphyrin dimers to human serum albumin (37o C, neutral pH, M−l) being 4 x 10* and 5 x 108 respectively. The temperature dependence (Dp and HSA, 22‐37o C) of the monomer binding showed the process to be entropy‐driven (δGo= ‐45 kJ mol−1; δSo=+146 kJ mol−1; δHo= 0 kJ mol−1). For the dimer binding, the enthalpy change was found to be highly temperature‐dependent implying continuous changes in the heat capacity of the system over the entire temperature range, the trend at the 37o C region fitting an entropy‐driven process. The monomer vs dimer differences in temperature dependence strongly support separate and independent binding sites for these species. Similar thermodynamics were determined for fatty‐acid carrying as well as for fatty‐acid free HSA, with mild quantitative (but not qualitative) shifts.