Inhibition of bone loss with surface-modulated, drug-loaded nanoparticles in an intraosseous model of prostate cancer.

Advanced-stage prostate cancer usually metastasizes to bone and is untreatable due to poor biodistribution of intravenously administered anticancer drugs to bone. In this study, we modulated the surface charge/composition of biodegradable nanoparticles (NPs) to sustain their blood circulation time and made them small enough to extravasate through the openings of the bone's sinusoidal capillaries and thus localize into marrow. NPs with a neutral surface charge, achieved by modulating the NP surface-associated emulsifier composition, were more effective at localizing to bone marrow than NPs with a cationic or anionic surface charge. These small neutral NPs (~150nm vs. the more usual ~320nm) were also ~7-fold more effective in localizing in bone marrow than large NPs. We hypothesized that NPs that effectively localize to marrow could improve NP-mediated anticancer drug delivery to sites of bone metastasis, thereby inhibiting cancer progression and preventing bone loss. In a PC-3M-luc cell-induced osteolytic intraosseous model of prostate cancer, these small neutral NPs demonstrated greater accumulation in bone within metastatic sites than in normal contralateral bone as well as co-localization with the tumor mass in marrow. Significantly, a single-dose intravenous administration of these small neutral NPs loaded with paclitaxel (PTX-NPs), but not anionic PTX-NPs, slowed the progression of bone metastasis. In addition, neutral PTX-NPs prevented bone loss, whereas animals treated with the rapid-release drug formulation Cremophor EL (PTX-CrEL) or saline (control) showed >50% bone loss. Neutral PTX-NPs did not cause acute toxicity, whereas animals treated with PTX-CrEL experienced weight loss. These results indicate that NPs with appropriate physical and sustained drug-release characteristics could be explored to treat bone metastasis, a significant clinical issue in prostate and other cancers.

Sustained Epigenetic Drug Delivery Depletes Cholesterol-Sphingomyelin Rafts from Resistant Breast Cancer Cells, Influencing Biophysical Characteristics of Membrane Lipids.

Cell-membrane lipid composition can greatly influence biophysical properties of cell membranes, affecting various cellular functions. We previously showed that lipid synthesis becomes altered in the membranes of resistant breast cancer cells (MCF-7/ADR); they form a more rigid, hydrophobic lipid monolayer than do sensitive cell membranes (MCF-7). These changes in membrane lipids of resistant cells, attributed to epigenetic aberration, significantly affected drug transport and endocytic function, thus impacting the efficacy of anticancer drugs. The present study's objective was to determine the effects of the epigenetic drug, 5-aza-2'-deoxycytidine (DAC), delivered in sustained-release nanogels (DAC-NGs), on the composition and biophysical properties of membrane lipids of resistant cells. Resistant and sensitive cells were treated with DAC in solution (DAC-sol) or DAC-NGs, and cell-membrane lipids were isolated and analyzed for lipid composition and biophysical properties. In resistant cells, we found increased formation of cholesterol-sphingomyelin (CHOL-SM) rafts with culturing time, whereas DAC treatment reduced their formation. In general, the effect of DAC-NGs was greater in changing the lipid composition than with DAC-sol. DAC treatment also caused a rise in levels of certain phospholipids and neutral lipids known to increase membrane fluidity, while reducing the levels of certain lipids known to increase membrane rigidity. Isotherm data showed increased lipid membrane fluidity following DAC treatment, attributed to decrease levels of CHOL-SM rafts (lamellar beta [Lß] structures or ordered gel) and a corresponding increase in lipids that form lamellar alpha-structures (Lα, liquid crystalline phase). Sensitive cells showed marginal or insignificant changes in lipid profile following DAC-treatment, suggesting that epigenetic changes affecting lipid biosynthesis are more specific to resistant cells. Since membrane fluidity plays a major role in drug transport and endocytic function, treatment of resistant cells with epigenetic drugs with altered lipid profile could facilitate anticancer drug transport to overcome acquired drug resistance in a combination therapy.

Biomechanics and thermodynamics of nanoparticle interactions with plasma and endosomal membrane lipids in cellular uptake and endosomal escape.

To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(D,L-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In conclusion, biomechanical interactions with membrane lipids are involved in cellular uptake and endosomal escape of NPs. Biophysical interaction studies could help us better understand the role of membrane lipids in cellular uptake and intracellular trafficking of NPs.

Heterogeneity in nanoparticles influences biodistribution and targeting.

AIM: A large fraction of the administered dose of nanoparticles (NPs) localizes into nontarget tissue, which could be due to the heterogeneous population of NPs. MATERIALS & METHODS: To investigate the impact of the above issue, we simultaneously tracked the biodistribution using optical imaging of two different sized poly(d,l-lactide co-glycolide) NPs, which also varied in their surface charge and texture, in a prostate tumor xenograft mouse model. RESULTS: Although formulated using the same polymer and emulsifier concentration, small NPs were neutral (S-neutral-NPs), whereas large NPs were anionic (L-anionic-NPs). Simultaneous injection of these NPs, representing heterogeneity, shows significantly different biodistribution. S-neutral-NPs demonstrated longer circulation time than L-anionic-NPs (t1/2 = 96 vs 13 min); accounted for 75% of total NPs accumulated in the tumor; and showed 13-fold greater tumor to liver signal intensity ratio than L-anionic-NPs. CONCLUSION: The data underscore the importance of formulating nanocarriers of specific properties to enhance their targeting efficacy.

Biophysics of cell membrane lipids in cancer drug resistance: Implications for drug transport and drug delivery with nanoparticles.

In this review, we focus on the biophysics of cell membrane lipids, particularly when cancers develop acquired drug resistance, and how biophysical changes in resistant cell membrane influence drug transport and nanoparticle-mediated drug delivery. Recent advances in membrane lipid research show the varied roles of lipids in regulating membrane P-glycoprotein function, membrane trafficking, apoptotic pathways, drug transport, and endocytic functions, particularly endocytosis, the primary mechanism of cellular uptake of nanoparticle-based drug delivery systems. Since acquired drug resistance alters lipid biosynthesis, understanding the role of lipids in cell membrane biophysics and its effect on drug transport is critical for developing effective therapeutic and drug delivery approaches to overcome drug resistance. Here we discuss novel strategies for (a) modulating the biophysical properties of membrane lipids of resistant cells to facilitate drug transport and regain endocytic function and (b) developing effective nanoparticles based on their biophysical interactions with membrane lipids to enhance drug delivery and overcome drug resistance.

Selective biophysical interactions of surface modified nanoparticles with cancer cell lipids improve tumor targeting and gene therapy.

Targeting gene- or drug-loaded nanoparticles (NPs) to tumors and ensuring their intratumoral retention after systemic administration remain key challenges to improving the efficacy of NP-based therapeutics. Here, we investigate a novel targeting approach that exploits changes in lipid metabolism and cell membrane biophysics that occur during malignancy. We hypothesized that modifications to the surface of NPs that preferentially increase their biophysical interaction with the membrane lipids of cancer cells will improve intratumoral retention and in vivo efficacy upon delivery of NPs loaded with a therapeutic gene. We have demonstrated that different surfactants, incorporated onto the NPs' surface, affect the biophysical interactions of NPs with the lipids of cancer cells and normal endothelial cells. NPs surface modified with didodecyldimethylammoniumbromide (DMAB) demonstrated greater interaction with cancer cell lipids, which was 6.7-fold greater than with unmodified NPs and 5.5-fold greater than with endothelial cell lipids. This correlated with increased uptake of DMAB-modified NPs with incubation time by cancer cells compared to other formulations of NPs and to uptake by endothelial cells. Upon systemic injection, DMAB-NPs demonstrated a 4.6-fold increase in tumor accumulation compared to unmodified NPs which also correlated to improved efficacy of p53 gene therapy. Characterization of the biophysical interactions between NPs and lipid membranes of tumors or other diseased tissues/organs may hold promise for engineering targeted delivery of therapeutics.

Epigenetic modulation of the biophysical properties of drug-resistant cell lipids to restore drug transport and endocytic functions.

In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane's biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug 5-aza-2'-deoxycytidine (decitabine) significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.

Drug resistance in breast cancer cells: biophysical characterization of and doxorubicin interactions with membrane lipids.

Understanding the role of lipids in drug transport is critical in cancer chemotherapy to overcome drug resistance. In this study, we isolated lipids from doxorubicin-sensitive (MCF-7) and -resistant (MCF-7/ADR) breast cancer cells to characterize the biophysical properties of membrane lipids (particularly lipid packing and membrane fluidity) and to understand the role of the interaction of cell membrane lipids with drug/nanocarrier on drug uptake and efficacy. Resistant cell membrane lipids showed significantly different composition and formed more condensed, less fluid monolayers than did lipids from sensitive cells. Doxorubicin, used as a model anticancer agent, showed a strong hydrophobic interaction with resistant cell membrane lipids but significantly less interaction, as well as a different pattern of interaction (i.e., ionic), with sensitive ones. The threshold intracellular doxorubicin concentration required to produce an antiproliferative effect was similar for both sensitive and resistant cell lines, suggesting that drug transport is a major barrier in determining drug efficacy in resistant cells. In addition to the biophysical characteristics of resistant cell membrane lipids, lipid-doxorubicin interactions appear to decrease intracellular drug transport via diffusion as the drug is trapped in the lipid bilayer. The rigid nature of resistant cell membranes also seems to influence endosomal functions that inhibit drug uptake when a liposomal formulation of doxorubicin is used. In conclusion, biophysical properties of resistant cell membrane lipids significantly influence drug transport, and hence drug efficacy. A better understanding of the mechanisms of cancer drug resistance is vital to developing more effective therapeutic interventions. In this regard, biophysical interaction studies with cell membrane lipids might be helpful to improve drug transport and efficacy through drug discovery and/or drug delivery approaches by overcoming the lipid barrier in resistant cells.

Biophysical interactions with model lipid membranes: applications in drug discovery and drug delivery.

The transport of drugs or drug delivery systems across the cell membrane is a complex biological process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug molecules. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid composition of cells and tissue in certain disease conditions may alter biophysical interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophysical interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery.

Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles.

The aim of the study was to test the hypothesis that the biophysical interactions of the trans-activating transcriptor (TAT) peptide-conjugated nanoparticles (NPs) with a model cell membrane could predict the cellular uptake of the encapsulated therapeutic agent. To test the above hypothesis, the biophysical interactions of ritonavir-loaded poly(l-lactide) nanoparticles (RNPs), conjugated to either a TAT peptide (TAT-RNPs) or a scrambled TAT peptide (sc-TAT-RNPs), were studied with an endothelial cell model membrane (EMM) using a Langmuir film balance, and the corresponding human vascular endothelial cells (HUVECs) were used to study the uptake of the encapsulated therapeutic. Biophysical interactions were determined from the changes in surface pressure (SP) of the EMM as a function of time following interaction with NPs, and the compression isotherm (pi-A) of the EMM lipid mixture in the presence of NPs. In addition, the EMMs were transferred onto a silicon substrate following interactions with NPs using the Langmuir-Schaeffer (LS) technique. The transferred LS films were imaged by atomic force microscopy (AFM) to determine the changes in lipid morphology and to characterize the NP-membrane interactions. TAT-RNPs showed an increase in SP of the EMM, which was dependent upon the amount of the peptide bound to NPs and the concentration of NPs, whereas sc-TAT-RNPs and RNPs did not show any significant change in SP. The isotherm experiment showed a shift toward higher mean molecular area (mmA) in the presence of TAT-RNPs, indicating their interactions with the lipids of the EMM, whereas sc-TAT-RNPs and RNPs did not show any significant change. The AFM images showed condensation of the lipids following interaction with TAT-RNPs, indicating their penetration into the EMM, whereas RNPs did not cause any change. Surface analysis and 3-D AFM images of the EMM further confirmed penetration of TAT-RNPs into the EMM, whereas RNPs were seen anchored loosely to the membrane, and were significantly less in number than TAT-RNPs. We speculate that hydrophobic tyrosine of the TAT that forms the NP-interface drives the initial interactions of TAT-RNPs with the EMM, followed by electrostatic interactions with the anionic phospholipids of the membrane. In the case of sc-TAT-RNPs, hydrophilic arginine forms the NP-interface that does not interact with the EMM, despite having the similar cationic charge on these NPs as TAT-RNPs. TAT peptide alone did not show any change in SP, suggesting that the interaction occurs when the peptide is conjugated to a carrier system. HUVECs showed higher uptake of the drug with TAT-RNPs as compared to that with sc-TAT-RNPs or RNPs, suggesting that the biophysical interactions of NPs with cell membrane lipids play a role in cellular internalization of NPs. In conclusion, TAT peptide sequence and the amount of TAT conjugated to NPs significantly affect the biophysical interactions of NPs with the EMM, and these interactions correlate with the cellular delivery of the encapsulated drug. Biophysical interactions with a model membrane thus could be effectively used in developing efficient functionalized nanocarrier systems for drug delivery applications.

Effect of molecular structure of cationic surfactants on biophysical interactions of surfactant-modified nanoparticles with a model membrane and cellular uptake.

The aim of this study was to test the hypothesis that the molecular structure of cationic surfactants at the nanoparticle (NP) interface influences the biophysical interactions of NPs with a model membrane and cellular uptake of NPs. Polystyrene NPs (surfactant-free, 130 nm) were modified with cationic surfactants. These surfactants were of either dichained (didodecyldimethylammonium bromide [DMAB]) or single-chained (cetyltrimethylammonium bromide [CTAB] and dodecyltrimethylammonium bromide [DTAB]) forms, with the latter two having different hydrophobic chain lengths. Biophysical interactions of these surfactant-modified NPs with an endothelial cell model membrane (EMM) were studied using a Langmuir film balance. Changes in surface pressure (SP) of EMM as a function of time following interaction with NPs and in the compression isotherm (pi-A) of the lipid mixture of EMM in the presence of NPs were analyzed. Langmuir-Schaeffer (LS) films, which are EMMs that have been transferred onto a suitable substrate, were imaged by atomic force microscopy (AFM), and the images were analyzed to determine the mechanisms of the NP-EMM interaction. DMAB-modified NPs showed a greater increase in SP and a shift toward higher mean molecular area (mmA) than CTAB- and DTAB-modified NPs, indicating stronger interactions of DMAB-modified NPs with the EMM. However, analysis of the AFM phase and height images of the LS films revealed that both DMAB- and CTAB-modified NPs interacted with the EMM but via different mechanisms: DMAB-modified NPs penetrated the EMM, thus explaining the increase in SP, whereas CTAB-modified NPs anchored onto the EMM's condensed lipid domains and hence did not cause any significant change in SP. Human umbilical vein endothelial cells showed greater uptake of DMAB- and CTAB-modified NPs than of DTAB-modified or unmodified NPs. We conclude that (i) the dichained and single-chained cationic surfactants on NPs have different mechanisms of interaction with the model membrane and that (ii) NPs that demonstrate greater biophysical interactions with the membrane also show greater cellular uptake. Biophysical interactions of NPs with a model membrane thus could be effectively used for developing nanocarriers with optimized surface properties for drug delivery and imaging applications.

Biophysical characterization of nanoparticle-endothelial model cell membrane interactions.

Understanding the biophysical interactions of nanoparticles (NPs) with cell membranes is critical for developing effective nanocarrier systems for drug delivery applications. We developed an endothelial model cell membrane (EMM) using a mixture of lipids and Langmuir balance to study its interaction with NPs. Polystyrene NPs of different surface chemistry and sizes were used as a model nanomaterial, and changes in the membrane's surface pressure (SP) were used as a parameter to monitor its interactions with NPs. Aminated NPs (60 nm) increased SP, plain NPs reduced it, and carboxylated NPs of the same size had no effect. However, smaller NPs (20 nm) increased SP irrespective of surface chemistry, and serum did not influence their SP effect, whereas it masked the effect of larger (>60 nm) plain and carboxylated but not that of aminated NPs. Membranes formed with a single phospholipid showed a different pattern of interactions with NPs than that with EMM, signifying the need of using a mixture of lipids representing the respective cells/tissue of interest for a model membrane. The particular effect of NP characteristics on SP, determined using atomic force microscopy and pi- A (surface pressure-area) isotherm, can be explained on the basis of whether the interaction results in condensation of phospholipids (increase in SP) or their displacement from the interface into the subphase (decrease in SP), causing destabilization of the membrane. We conclude that NP characteristics significantly influence biophysical interactions with the membrane. Further, the molecular mechanism(s) of nanoparticle interactions with model membranes can be effectively used for optimizing the characteristics of nanomaterials for particular biological applications.

Water surface covering of fluorinated amphiphilic triblock copolymers: surface pressure-area and X-ray reflectivity investigations.

Monolayers of ABA amphiphilic triblock block copolymers are studied using surface pressure-area and X-ray reflectivity (XR) measurements. The triblock copolymers are composed of long poly(ethylene oxide) (PEO) middle blocks with poly((perfluorohexyl)ethyl methacrylate) (PFMA) end blocks. The surface pressure-area isotherms of water-insoluble species show two pseudoplateaus. The plateau at low surface pressure is consistent with the pseudoplateau observed for PEO copolymers in the literature. The plateau in the brush region can be assigned to the horizontal to vertical rearrangement of whole PFMA chains at the air-water interface, which was followed by XR measurements. For water-soluble species with a very low amount of PFMA no (significant) second pseudoplateau and no enrichment of PFMA at the air-water interface were observed.