Combined, yet separate: cocktails of carriers (not drugs) for actinium-225 α-particle therapy of solid tumors expressing moderate-to-low levels of targetable markers.

Alpha-particle radionuclide-antibody conjugates are being clinically evaluated against solid tumors even when they moderately express the targeted markers. At this limit of lower tumor-absorbed doses, to maintain efficacy, the few(er) intratumorally delivered alpha-particles need to traverse/hit as many different cancer cells as possible. We complement antibody-radioconjugate therapies with a separate nanocarrier delivering a fraction of the same total injected radioactivity to tumor regions geographically different than those affected by targeting antibodies; these carrier-cocktails collectively distribute the alpha-particle emitters better. METHODS: The efficacy of actinium-225 delivered by our carrier-cocktails was assessed in vitro and on mice with orthotopic MDA-MB-436 and/or MDA-MB-231 triple-negative breast cancers and/or an ectopic BxPC3 pancreatic cancer. Cells/tumors were chosen to express low-to-moderate levels of HER1, as model antibody-targeted marker. RESULTS: Independent of cell line, antibody-radioconjugates were most lethal on cell monolayers. On spheroids, with radii greater than alpha-particles' range, carrier-cocktails improved killing efficacy (p < 0.0500). Treatment with carrier-cocktails decreased the MDA-MB-436 and MDA-MB-231 orthotopic tumor volumes by 73.7% and 72.1%, respectively, relative to treatment with antibody-radioconjugates alone, at same total injected radioactivity; these carrier-cocktails completely eliminated formation of spontaneous metastases vs. 50% and 25% elimination in mice treated with antibody-radioconjugates alone. In BxPC3 tumor-bearing mice, carrier-cocktails increased the median survival to 25-26 days (in male-female animals) vs. 20-21 days of mice treated with antibody-radioconjugates alone (vs. 17 days for non-treated animals). Survival with carrier-cocktail radiotherapy was further prolonged by pre-injecting low-dose, standard-of-care, gemcitabine (p = 0.0390). CONCLUSION: Tumor-agnostic carrier-cocktails significantly enhance the therapeutic efficacy of existing alpha-particle radionuclide-antibody treatments.

The Rate of Cisplatin Dosing Affects the Resistance and Metastatic Potential of Triple Negative Breast Cancer Cells, Independent of Hypoxia.

To best control tumor growth and/or metastasis in triple negative breast cancer (TNBC), it may be useful to understand the effect(s) of chemotherapy delivery (i.e., the rate and pattern of exposure to the drug) on cell sub-populations that have experienced different levels of hypoxia (and/or acidosis). In this spirit, MDA-MB-231 TNBC cells, and their hypoxia-reporter counterparts, were characterized for their sensitivity to cisplatin. When in the form of multicellular spheroids, that capture the diffusion-limited transport that generates hypoxic and acidic subregions within the avascular areas of solid tumors, the effects of the rate and pattern of exposure to cisplatin on cell viability and motility/migration potential were evaluated for each cell sub-population. We demonstrated that cell sensitivity to cisplatin was not dependent on acidosis, but cell resistance increased with exposure to hypoxia. In spheroids, the increase of the rates of cell exposure to cisplatin, at a constant cumulative dose, increased sensitivity to chemotherapy and lowered the cells' metastatic potential, even for cells that had experienced hypoxia. This effect was also shown to be caused by nanocarriers engineered to quickly release cisplatin which deeply penetrated the spheroid interstitium, resulting in the fast and uniform exposure of the TNBC tumors to the agent. This rate and dosing-controlled model may effectively limit growth and/or metastasis, independent of hypoxia. This mode of chemotherapy delivery can be enabled by engineered nanocarriers.

Predicting response of micrometastases with MIRDcell V3: proof of principle with 225Ac-DOTA encapsulating liposomes that produce different activity distributions in tumor spheroids.

PURPOSE: The spatial distribution of radiopharmaceuticals within multicellular clusters is known to have a significant effect on their biological response. Most therapeutic radiopharmaceuticals distribute nonuniformly in tissues which makes predicting responses of micrometastases challenging. The work presented here analyzes published temporally dependent nonuniform activity distributions within tumor spheroids treated with actinium-225-DOTA encapsulating liposomes (225Ac-liposomes) and uses these data in MIRDcell V3.11 to calculate absorbed dose distributions and predict biological response. The predicted responses are compared with experimental responses. METHODS: Four types of liposomes were prepared having membranes with different combinations of release (R) and adhesion (A) properties. The combinations were R-A-, R-A+, R+A-, and R+A+. These afford different penetrating properties into tissue. The liposomes were loaded with either carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) or 225Ac. MDA-MB-231 spheroids were treated with the CFDA-SE-liposomes, harvested at different times, and the time-integrated CFDA-SE concentration at each radial position within the spheroid was determined. This was translated into mean 225Ac decays/cell versus radial position, uploaded to MIRDcell, and the surviving fraction of cells in spherical multicellular clusters was simulated. The MIRDcell-predicted surviving fractions were compared with experimental fractional-outgrowths of the spheroids following treatment with 225Ac-liposomes. RESULTS: The biological responses of the multicellular clusters treated with 225Ac-liposomes with physicochemical properties R+A+, R-A+, and R-A- were predicted by MIRDcell with statistically significant accuracy. The prediction for R+A- was not predicted accurately. CONCLUSION: In most instances, MIRDcell predicts responses of spheroids treated with 225Ac-liposomes that result in different tissue-penetrating profiles of the delivered radionuclides.

Two diverse carriers are better than one: A case study in α-particle therapy for prostate specific membrane antigen-expressing prostate cancers.

Partial and/or heterogeneous irradiation of established (i.e., large, vascularized) tumors by α-particles that exhibit only a 4-5 cell-diameter range in tissue, limits the therapeutic effect, since regions not being hit by the high energy α-particles are likely not to be killed. This study aims to mechanistically understand a delivery strategy to uniformly distribute α-particles within established solid tumors by simultaneously delivering the same α-particle emitter by two diverse carriers, each killing a different region of the tumor: (1) the cancer-agnostic, but also tumor-responsive, liposomes engineered to best irradiate tumor regions far from the vasculature, and (2) a separately administered, antibody, targeting any cancer-cell's surface marker, to best irradiate the tumor perivascular regions. We demonstrate that on a prostate specific membrane antigen (PSMA)-expressing prostate cancer xenograft mouse model, for the same total injected radioactivity of the α-particle emitter Actinium-225, any radioactivity split ratio between the two carriers resulted in better tumor growth inhibition compared to the tumor inhibition when the total radioactivity was delivered by any of the two carriers alone. This finding was due to more uniform tumor irradiation for the same total injected radioactivity. The killing efficacy was improved even though the tumor-absorbed dose delivered by the combined carriers was lower than the tumor-absorbed dose delivered by the antibody alone. Studies on spheroids with different receptor-expression, used as surrogates of the tumors' avascular regions, demonstrated that our delivery strategy is valid even for as low as 1+ (ImmunoHistoChemistry score) PSMA-levels. The findings presented herein may hold clinical promise for those established tumors not being effectively eradicated by current α-particle radiotherapies.

Combination of Carriers with Complementary Intratumoral Microdistributions of Delivered α-Particles May Realize the Promise for 225Ac in Large, Solid Tumors.

α-particle radiotherapy has already been shown to be impervious to most resistance mechanisms. However, in established (i.e., large, vascularized) soft-tissue lesions, the diffusion-limited penetration depths of radiolabeled antibodies or nanocarriers (≤50-80 µm) combined with the short range of α-particles (4-5 cell diameters) may result in only partial tumor irradiation, potentially limiting treatment efficacy. To address this challenge, we combined carriers with complementary intratumoral microdistributions of the delivered α-particles. We used the α-particle generator 225Ac, and we combined a tumor-responsive liposome (which, on tumor uptake, releases into the interstitium a highly diffusing form of its radioactive payload [225Ac-DOTA], potentially penetrating the deeper parts of tumors where antibodies do not reach) with a separately administered, less-penetrating radiolabeled antibody (irradiating the tumor perivascular regions where liposome contents clear too quickly). Methods: In a murine model with orthotopic human epidermal growth factor receptor 2-positive BT474 breast cancer xenografts, the biodistributions of each carrier were evaluated, and the control of tumor growth was monitored after administration of the same total radioactivity of 225Ac delivered by the 225Ac-DOTA-encapsulating liposomes, by the 225Ac-DOTA-SCN--labeled trastuzumab, and by both carriers at equally split radioactivities. Results: Tumor growth was significantly more inhibited when the same total injected radioactivity was divided between the 2 separate carriers than when delivered by either of the carriers alone. The combined carriers enabled more uniform intratumoral microdistributions of α-particles, at a tumor dose that was lower than the dose delivered by the antibody alone. Conclusion: This strategy demonstrates that more uniform microdistributions of the delivered α-particles within established solid tumors improve efficacy even at lower tumor doses. Augmentation of antibody-targeted α-particle therapies with tumor-responsive liposomes may address partial tumor irradiation, improving therapeutic effects.

Growth Inhibition of Triple-Negative Breast Cancer: The Role of Spatiotemporal Delivery of Neoadjuvant Doxorubicin and Cisplatin.

Combinations of platinum-based compounds with doxorubicin in free and/or in liposomal form for improved safety are currently being evaluated in the neoadjuvant setting on patients with advanced triple-negative breast cancer (TNBC). However, TNBC may likely be driven by chemotherapy-resistant cells. Additionally, established TNBC tumors may also exhibit diffusion-limited transport, resulting in heterogeneous intratumoral delivery of the administered therapeutics; this limits therapeutic efficacy in vivo. We studied TNBC cells with variable chemosensitivities, in the absence (on monolayers) and presence (in 3D multicellular spheroids) of transport barriers; we compared the combined killing effect of free doxorubicin and free cisplatin to the killing effect (1) of conventional liposomal forms of the two chemotherapeutics, and (2) of tumor-responsive lipid nanoparticles (NP), specifically engineered to result in more uniform spatiotemporal microdistributions of the agents within solid tumors. This was enabled by the NP properties of interstitial release, cell binding/internalization, and/or adhesion to the tumors' extracellular matrix. The synergistic cell kill by combinations of the agents (in all forms), compared to the killing effect of each agent alone, was validated on monolayers of cells. Especially for spheroids formed by cells exhibiting resistance to doxorubicin combination treatments with both agents in free and/or in tumor-responsive NP-forms were comparably effective; we not only observed greater inhibition of outgrowth compared to the single agent(s) but also compared to the conventional liposome forms of the combined agents. We correlated this finding to more uniform spatiotemporal microdistributions of agents by the tumor-responsive NP. Our study shows that combinations of NP with properties specifically optimized to improve the spatiotemporal uniformity of the delivery of their corresponding therapeutic cargo can improve treatment efficacy while keeping favorable safety profiles.

Transport-driven engineering of liposomes for delivery of α-particle radiotherapy to solid tumors: effect on inhibition of tumor progression and onset delay of spontaneous metastases.

PURPOSE: Highly cytotoxic α-particle radiotherapy delivered by tumor-selective nanocarriers is evaluated on metastatic Triple Negative Breast Cancer (TNBC). On vascularized tumors, the limited penetration of nanocarriers (<50-80 µm) combined with the short range of α-particles (40-100 µm) may, however, result in only partial tumor irradiation, compromising efficacy. Utilizing the α-particle emitter Actinium-225 (225Ac), we studied how the therapeutic potential of a general delivery strategy using nanometer-sized engineered liposomes was affected by two key transport-driven properties: (1) the release from liposomes, when in the tumor interstitium, of the highly diffusing 225Ac-DOTA that improves the uniformity of tumor irradiation by α-particles and (2) the adhesion of liposomes on the tumors' ECM that increases liposomes' time-integrated concentrations within tumors and, therefore, the tumor-delivered radioactivities. METHODS: On an orthotopic MDA-MB-231 TNBC murine model forming spontaneous metastases, we evaluated the maximum tolerated dose (MTD), biodistributions, and control of tumor growth and/or spreading after administration of 225Ac-DOTA-encapsulating liposomes, with different combinations of the two transport-driven properties. RESULTS: At 83% of MTD, 225Ac-DOTA-encapsulating liposomes with both properties (1) eliminated formation of spontaneous metastases and (2) best inhibited the progression of orthotopic xenografts, compared to liposomes lacking one or both properties. These findings were primarily affected by the extent of uniformity of the intratumoral microdistributions of 225Ac followed by the overall tumor uptake of radioactivity. At the MTD, long-term toxicities were not detected 9.5 months post administration. CONCLUSION: Our findings demonstrate the potential of a general, transport-driven strategy enabling more uniform and prolonged solid tumor irradiation by α-particles without cell-specific targeting.

Daptomycin-Induced Lipid Phases on Model Lipid Bilayers: Effect of Lipid Type and of Lipid Leaflet Order on Membrane Permeability.

Daptomycin's bacterial membrane activity is partly due to the defects of lipid-packing at the boundaries of daptomycin-induced, separated lipid phases, which are rich in phosphatidylglycerol (PG). On model membranes, the permeability of phase boundaries is strongly dependent on the extent of saturation of the lipid acyl tails, which affect the lipids' ability to pack within these boundaries, and on the cross-leaflet registration of these boundaries. Using vesicles with asymmetric lipid leaflet compositions, we evaluated the role of headgroup type and/or extent of acyl-tail saturation on daptomycin-induced membrane permeability. We demonstrate that the release rates of vesicle-encapsulated contents scales with the total length of daptomycin-induced, PG-rich phase boundaries. On the outer leaflet, lipids with PG-headgroups (in contact with daptomycin) were a necessary condition, but they still were not adequate for release. Increased membrane permeability was observed only when inner leaflet lipids had saturated acyl tails; we postulate that the latter may have enabled the recruitment, by the outer leaflet daptomycin-induced phases, of inner leaflet lipids in cross-registered phases with boundaries of defective packing that spanned the bilayer. These findings provide insights on the potential role of lipids as a whole (headgroup and acyl tails) and of lipid leaflet order on the boundaries of daptomycin-induced separated lipid phases in model membranes.

Growth of Metastatic Triple-Negative Breast Cancer Is Inhibited by Deep Tumor-Penetrating and Slow Tumor-Clearing Chemotherapy: The Case of Tumor-Adhering Liposomes with Interstitial Drug Release.

The poor prognosis of triple-negative breast cancer (TNBC) is attributed largely to the lack of tumor-selective therapeutic modalities that effectively deliver lethal doses at the sites of metastatic disease. Tumor-selective drug delivery strategies that aim to improve uniformity in intratumoral drug microdistributions and to prolong exposure of these cancer cells to delivered therapeutics may improve therapeutic efficacy against established TNBC metastases. In this study, we present lipid carriers for selective (due to their nanometer size) tumor delivery, which are loaded with cisplatin and designed to exhibit the following properties when in the tumor interstitium: (1) interstitial drug release (for deeper tumor penetration of cisplatin) and/or (2) intratumoral/interstitial adhesion of the carriers to tumors' extracellular matrix (ECM)-not accompanied by cell internalization-for delayed tumor clearance of carriers prolonging cancer cell exposure to the cisplatin being released. We show that on large multicellular spheroids, used as surrogates of avascular solid tumor regions, greater growth inhibition was strongly correlated with spatially more uniform drug concentrations (due to interstitial drug release) and with longer exposure to the released drug (i.e., higher time-integrated drug concentrations enabled by slow clearing of adhesive nanoparticles). Lipid nanoparticles with both the release and adhesion properties were the most effective, followed by nanoparticles with only the releasing property and then by nanoparticles with only the adhering property. In vivo, cisplatin-loaded nanoparticles with releasing and/or adhering properties significantly inhibited the growth of spontaneous TNBC metastases compared to conventional liposomal cisplatin, and the efficacy of different property combinations followed the same trends as in spheroids. This study demonstrates the therapeutic potential of a general strategy to bypass treatment limitations of established TNBC metastases due to the lack of cell-targeting markers: aiming to optimize the temporal intratumoral drug microdistributions for more uniform and prolonged drug exposure.

Topical inhibition of PUMA signaling mitigates radiation injury.

Radiation therapy is an effective treatment strategy for many types of cancer but is limited by its side effects on normal tissues, particularly the skin, where persistent and progressive fibrotic changes occur and can impair wound healing. In this study, we attempted to mitigate the effects of irradiation on skin using a novel transcutaneous topical delivery system to locally inhibit p53 up-regulated modulator of apoptosis (PUMA) gene expression with small interfering RNA (siRNA). In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated. Prior to irradiation, PUMA and nonsense siRNA were applied via a novel hydrogel formulation to dorsal skin and reapplied weekly. Skin was harvested at multiple time points to evaluate dermal siRNA penetration, mRNA expression, protein expression, dermal thickness, subcutaneous fat, stiffness, vascular hypertrophy, SCAR index, and reactive oxygen species (ROS) generation. Murine skin treated with topical PUMA siRNA via optimized hydrogel formulation demonstrated effective PUMA inhibition in irradiated tissue at 3-4 days. Tissue stiffness, dermal thickness, vascular hypertrophy, SCAR index, ROS levels, and mRNA levels of MnSOD and TGF-ß were all significantly reduced with siPUMA treatment compared to nonsense controls. Subcutaneous fat area was significantly increased, and levels of SMAD3 and Phospho-SMAD3 expression were unchanged. These results show that PUMA expression can be effectively silenced in vivo using a novel hydrogel lipoplex topical delivery system. Moreover, cutaneous PUMA inhibition mitigates radiation induced changes in tissue character, restoring a near-normal phenotype independent of SMAD3 signaling.

Kinetic Analysis of Nanostructures Formed by Enzyme-Instructed Intracellular Assemblies against Cancer Cells.

Recent studies have demonstrated that enzyme-instructed self-assembly (EISA) in extra- or intracellular environments can serve as a multistep process for controlling cell fate. There is little knowledge, however, about the kinetics of EISA in the complex environments in or around cells. Here, we design and synthesize three dipeptidic precursors (ld-1-SO3, dl-1-SO3, dd-1-SO3), consisting of diphenylalanine (l-Phe-d-Phe, d-Phe-l-Phe, d-Phe-d-Phe, respectively) as the backbone, which are capped by 2-(naphthalen-2-yl)acetic acid at the N-terminal and by 2-(4-(2-aminoethoxy)-4-oxobutanamido)ethane-1-sulfonic acid at the C-terminal. On hydrolysis by carboxylesterases (CES), these precursors result in hydrogelators, which self-assemble in water at different rates. Whereas all three precursors selectively kill cancer cells, especially high-grade serous ovarian carcinoma cells, by undergoing intracellular EISA, dl-1-SO3 and dd-1-SO3 exhibit the lowest and the highest activities, respectively, against the cancer cells. This trend inversely correlates with the rates of converting the precursors to the hydrogelators in phosphate-buffered saline. Because CES exists both extra- and intracellularly, we use kinetic modeling to analyze the kinetics of EISA inside cells and to calculate the cytotoxicity of each precursor for killing cancer cells. Our results indicate that (i) the stereochemistry of the precursors affects the morphology of the nanostructures formed by the hydrogelators, as well as the rate of enzymatic conversion; (ii) decreased extracellular hydrolysis of precursors favors intracellular EISA inside the cells; (iii) the inherent features ( e.g., self-assembling ability and morphology) of the EISA molecules largely dictate the cytotoxicity of intracellular EISA. As the kinetic analysis of intracellular EISA, this work elucidates how the stereochemistry modulates EISA in the complex extra- and/or intracellular environment for developing anticancer molecular processes. Moreover, it provides insights for understanding the kinetics and cytotoxicity of aggregates of aberrant proteins or peptides formed inside and outside cells.

Targeted and Nontargeted α-Particle Therapies.

α-Particle irradiation of cancerous tissue is increasingly recognized as a potent therapeutic option. We briefly review the physics, radiobiology, and dosimetry of α-particle emitters, as well as the distinguishing features that make them unique for radiopharmaceutical therapy. We also review the emerging clinical role of α-particle therapy in managing cancer and recent studies on in vitro and preclinical α-particle therapy delivered by antibodies, other small molecules, and nanometer-sized particles. In addition to their unique radiopharmaceutical characteristics, the increased availability and improved radiochemistry of α-particle radionuclides have contributed to the growing recent interest in α-particle radiotherapy. Targeted therapy strategies have presented novel possibilities for the use of α-particles in the treatment of cancer. Clinical experience has already demonstrated the safe and effective use of α-particle emitters as potent tumor-selective drugs for the treatment of leukemia and metastatic disease.

Clustered versus Uniform Display of GALA-Peptides on Carrier Nanoparticles: Enhancing the Permeation of Noncharged Fluid Lipid Membranes.

GALA-peptide is a random coil in neutral pH; in acidic pH, it becomes an amphipathic α-helix that aggregates in solution, possibly via its hydrophobic facet that runs along the helix's long axis. In the presence of fluid lipid membranes, the GALA-helix exhibits membrane-active properties that originate from the same hydrophobic facet; these properties make GALA a candidate for inclusion in drug delivery systems requiring permeation of the endosomal membrane to enable drug escape into the cytoplasm. Previous work has shown that the uniform functionalization of carrier nanoparticles with GALA-peptides improved their membrane activity and enhanced the endosomal escape of delivered therapeutics. The present study aims to evaluate the potential role of altering membrane activity via cluster-displayed GALA-peptides (for higher local valency) on the surface of carrier nanoparticles. The presentation of GALA-peptides on carrier nanoparticles was also designed to be pH-dependent. The peptide display on the surface of the carrier nanoparticles was uniform in neutral pH; in the acidic endosomal pH, the surface of nanocarriers formed domains (patches) with high local densities of GALA-peptides. The interactions between GALA-functionalized carrier nanoparticles and target lipid vesicles, utilized as endosome membrane surrogates that were used to primarily capture the fluid nature of these membranes, were studied as a function of pH. At endosomal pH values, ranging from 5.5 to 5.0, the greatest permeability of target membranes was induced by nanocarriers with clustered rather than uniformly displayed GALA. This enhancing effect had an optimum; at even more acidic pH values, too close an approximation of GALA-peptides residing within the same patches resulted in preferential intrapatch peptide interactions rather than interactions with the apposing target lipid membranes. This behavior could have the same physicochemical origin as the aforementioned GALA-peptide aggregation, observed in solution with decreasing pH at increasing peptide concentrations. The findings of this study support the potential of utilizing the clustered display of GALA-peptides on carrier nanoparticles to increase the permeation of fluid membranes used herein to capture this critical physical property of endosomal membranes and therefore to ultimately improve the endosomal escape of delivered therapeutics, enhancing therapeutic efficacy.

Alpha-particle radiotherapy: For large solid tumors diffusion trumps targeting.

Diffusion limitations on the penetration of nanocarriers in solid tumors hamper their therapeutic use when labeled with α-particle emitters. This is mostly due to the α-particles' relatively short range (≤100 µm) resulting in partial tumor irradiation and limited killing. To utilize the high therapeutic potential of α-particles against solid tumors, we designed non-targeted, non-internalizing nanometer-sized tunable carriers (pH-tunable liposomes) that are triggered to release, within the slightly acidic tumor interstitium, highly-diffusive forms of the encapsulated α-particle generator Actinium-225 (225Ac) resulting in more homogeneous distributions of the α-particle emitters, improving uniformity in tumor irradiation and increasing killing efficacies. On large multicellular spheroids (400 µm-in-diameter), used as surrogates of the avascular areas of solid tumors, interstitially-releasing liposomes resulted in best growth control independent of HER2 expression followed in performance by (a) the HER2-targeting radiolabeled antibody or (b) the non-responsive liposomes. In an orthotopic human HER2-negative mouse model, interstitially-releasing 225Ac-loaded liposomes resulted in the longest overall and median survival. This study demonstrates the therapeutic potential of a general strategy to bypass the diffusion-limited transport of radionuclide carriers in solid tumors enabling interstitial release from non-internalizing nanocarriers of highly-diffusing and deeper tumor-penetrating molecular forms of α-particle emitters, independent of cell-targeting.

Interstitial Release of Cisplatin from Triggerable Liposomes Enhances Efficacy against Triple Negative Breast Cancer Solid Tumor Analogues.

Liposomal cisplatin, a promising triple negative breast cancer treatment modality, has been shown to decrease toxicities associated with cisplatin's free agent form. However, the heterogeneous intratumoral distributions of the liposomes themselves, combined with limited release of cisplatin from them contribute to limited penetration of cisplatin within tumors reducing efficacy. This study uses pH-responsive liposomes designed to release cisplatin within the acidic tumor interstitium (7.0 > pH ≥ 6.0) with a dual aim (1) to improve the penetration of the free drug within tumors on the assumption of greater diffusivities based on the free drug's much smaller size than its carrier's size and (2) to increase the availability of the free agent near cancer cells deep into the tumor. On cell monolayers treated with pH-releasing liposomal cisplatin, acidification of the extracellular solution resulted in decreased LD50 values, which were significantly lower than the LD50 values for non-pH-releasing liposomal cisplatin. In multicellular spheroids with acidic interstitia, pH-releasing liposomal cisplatin significantly decreased spheroid volumes relative to non-pH-releasing liposomal cisplatin. Improved efficacy was correlated with increased spheroid penetration of a fluorescent cisplatin surrogate. These findings demonstrate that interstitial release of cisplatin by pH-responsive liposomes may improve the intratumoral distributions of the free drug enhancing efficacy.

Sticky Patches on Lipid Nanoparticles Enable the Selective Targeting and Killing of Untargetable Cancer Cells.

Effective targeting by uniformly functionalized nanoparticles is limited to cancer cells expressing at least two copies of targeted receptors per nanoparticle footprint (approximately ≥2 × 10(5) receptor copies per cell); such a receptor density supports the required multivalent interaction between the neighboring receptors and the ligands from a single nanoparticle. To enable selective targeting below this receptor density, ligands on the surface of lipid vesicles were displayed in clusters that were designed to form at the acidic pH of the tumor interstitium. Vesicles with clustered HER2-targeting peptides within such sticky patches (sticky vesicles) were compared to uniformly functionalized vesicles. On HER2-negative breast cancer cells MDA-MB-231 and MCF7 {expressing (8.3 ± 0.8) × 10(4) and (5.4 ± 0.9) × 10(4) HER2 copies per cell, respectively}, only the sticky vesicles exhibited detectable specific targeting (KD ≈ 49-69 nM); dissociation (0.005-0.009 min(-1)) and endocytosis rates (0.024-0.026 min(-1)) were independent of HER2 expression for these cells. MDA-MB-231 and MCF7 were killed only by sticky vesicles encapsulating doxorubicin (32-40% viability) or α-particle emitter (225)Ac (39-58% viability) and were not affected by uniformly functionalized vesicles (>80% viability). Toxicities on cardiomyocytes and normal breast cells (expressing HER2 at considerably lower but not insignificant levels) were not observed, suggesting the potential of tunable clustered ligand display for the selective killing of cancer cells with low receptor densities.

Nanoconjugation of PSMA-Targeting Ligands Enhances Perinuclear Localization and Improves Efficacy of Delivered Alpha-Particle Emitters against Tumor Endothelial Analogues.

This study aims to evaluate the effect on killing efficacy of the intracellular trafficking patterns of α-particle emitters by using different radionuclide carriers in the setting of targeted antivascular α-radiotherapy. Nanocarriers (lipid vesicles) targeted to the prostate-specific membrane antigen (PSMA), which is unique to human neovasculature for a variety of solid tumors, were loaded with the α-particle generator actinium-225 and were compared with a PSMA-targeted radiolabeled antibody. Actinium-225 emits a total of four α-particles per decay, providing highly lethal and localized irradiation of targeted cells with minimal exposure to surrounding healthy tissues. Lipid vesicles were derivatized with two types of PSMA-targeting ligands: a fully human PSMA antibody (mAb) and a urea-based, low-molecular-weight agent. Target selectivity and extent of internalization were evaluated on monolayers of human endothelial cells (HUVEC) induced to express PSMA in static incubation conditions and in a flow field. Both types of radiolabeled PSMA-targeted vesicles exhibit similar killing efficacy, which is greater than the efficacy of the radiolabeled control mAb when compared on the basis of delivered radioactivity per cell. Fluorescence confocal microscopy demonstrates that targeted vesicles localize closer to the nucleus, unlike antibodies which localize near the plasma membrane. In addition, targeted vesicles cause larger numbers of dsDNAs per nucleus of treated cells compared with the radiolabeled mAb. These findings demonstrate that radionuclide carriers, such as PSMA-targeted lipid-nanocarriers, which localize close to the nucleus, increase the probability of α-particle trajectories crossing the nuclei, and, therefore, enhance the killing efficacy of α-particle emitters.

Phase separation behavior of mixed lipid systems at neutral and low pH: coarse-grained simulations with DMD/LIME.

We extend LIME, an intermediate resolution, implicit solvent model for phospholipids previously used in discontinuous molecular dynamics simulations of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer formation at 325 K, to the description of the geometry and energetics of 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS) and 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine (21PC) and mixtures thereof at both neutral and low pH at 310 K. A multiscale modeling approach is used to calculate the LIME parameters from atomistic simulation data on a mixed DPPC/DSPS system at different pH values. In the model, 17 coarse-grained sites represent DSPS and 18 coarse-grained sites represent 21PC. Each of these coarse-grained sites is classified as 1 of 9 types. LIME/DMD simulations of equimolar bilayers show the following: (1) 21PC/DSPS bilayers with and without surface area restrictions separate faster at low pH than at neutral pH, (2) 21PC/DSPS systems separate at approximately the same rate regardless of whether they are subjected to surface area restrictions, and (3) bilayers with a molar ratio of 9:1 (21PC:DSPS) phase separate to form heterogeneous domains faster at low pH than at neutral pH. Our results are consistent with experimental findings of Sofou and co-workers (Bandekar et al. Mol. Pharmaceutics, 2013, 10, 152-160; Karve et al. Biomaterials, 2010, 31, 4409-4416) that more doxorubicin is released from 21PC/DSPS liposomes at low pH than at neutral pH, presumably because greater phase separation is achieved at low pH than at neutral pH. These are the first molecular-level simulations of the phase separation in mixed lipid bilayers induced by a change in pH.

Liposome-based approaches for delivery of mainstream chemotherapeutics: preparation methods, liposome designs, therapeutic efficacy.

We review liposome-based delivery approaches that aim to address toxicities and to improve the therapeutic efficacy of mainstream chemotherapeutics, namely, doxorubicin, paclitaxel, and cisplatin. A brief review of the biomolecular mechanism(s) of action of these agents is followed by a description of characteristic examples of therapeutic approaches and of liposome membrane designs. Short reports on clinical studies are also included when applicable. The technical issues of different loading/encapsulation methods of these agents into liposomes are also discussed in terms of the physicochemical properties of both the agents themselves and of the lipid-based self-assemblies.