Photo-triggerable liposomes based on lipid-porphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Magnetism-mediated targeting hyperthermia-immunotherapy in "cold" tumor with CSF1R inhibitor.

Background: Immunotherapy has profoundly changed the landscape of cancer management and represented the most significant breakthrough. Yet, it is a formidable challenge that the majority of cancers - the so-called "cold" tumors - poorly respond to immunotherapy. To find a general immunoregulatory modality that can be applied to a broad spectrum of cancers is an urgent need. Methods: Magnetic hyperthermia (MHT) possesses promise in cancer therapy. We develop a safe and effective therapeutic strategy by using magnetism-mediated targeting MHT-immunotherapy in "cold" colon cancer. A magnetic liposomal system modified with cell-penetrating TAT peptide was developed for targeted delivery of a CSF1R inhibitor (BLZ945), which can block the CSF1-CSF1R pathway and reduce M2 macrophages. The targeted delivery strategy is characterized by its magnetic navigation and TAT-promoting intratumoral penetration. Results: The liposomes (termed TAT-BLZmlips) can induce ICD and cause excessive CRT exposure on the cell surface, which transmits an "eat-me" signal to DCs to elicit immunity. The combination of MHT and BLZ945 can repolarize M2 macrophages in the tumor microenvironment to relieve immunosuppression, normalize the tumor blood vessels, and promote T-lymphocyte infiltration. The antitumor effector CD8+ T cells were increased after treatment. Conclusion: This work demonstrated that TAT-BLZmlips with magnetic navigation and MHT can remodel tumor microenvironment and activate immune responses and memory, thus inhibiting tumor growth and recurrence.

Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell.

Artificial systems capable of self-sustained movement with self-sufficient energy are of high interest with respect to the development of many challenging applications, including medical treatments, but also technical applications. The bottom-up assembly of such systems in the context of synthetic biology is still a challenging task. In this work, we demonstrate the biocompatibility and efficiency of an artificial light-driven energy module and a motility functional unit by integrating light-switchable photosynthetic vesicles with demembranated flagella. The flagellar propulsion is coupled to the beating frequency, and dynamic ATP synthesis in response to illumination allows us to control beating frequency of flagella in a light-dependent manner. In addition, we verified the functionality of light-powered synthetic vesicles in in vitro motility assays by encapsulating microtubules assembled with force-generating kinesin-1 motors and the energy module to investigate the dynamics of a contractile filamentous network in cell-like compartments by optical stimulation. Integration of this photosynthetic system with various biological building blocks such as cytoskeletal filaments and molecular motors may contribute to the bottom-up synthesis of artificial cells that are able to undergo motor-driven morphological deformations and exhibit directional motion in a light-controllable fashion.

Ultrasound Controlled Anti-Inflammatory Polarization of Platelet Decorated Microglia for Targeted Ischemic Stroke Therapy.

Stroke is a lethal cerebral disease with severe sequelae and high mortality. Microglia, the main immune cell in the cerebrum, possess therapeutic potential for strokes as its specific anti-inflammatory phenotype can reduce inflammation and promote neuron regeneration. However, the on-demand anti-inflammatory polarization of microglia at the stroke site is uncontrollable for therapeutic application. Here, we develop a platelet hybrid microglia platform which can specifically polarize to the anti-inflammatory phenotype by ultrasound irradiation for targeted cerebrum repair after stroke. The engineered microglia have strong adherence to the injured cerebral vessels with platelet membrane fusion and realize on-demand anti-inflammatory polarization with ultrasound-responsive IL-4 liposome decoration. The intravenously injected microglia platform showed anti-inflammatory polarization at the stroke site with insonation, and accelerated the M2-type polarization of endogenous microglia for long-term stroke recovery. Satisfied prognoses were achieved with reduced apoptosis, promoted neurogenesis, and functional recovery, indicating the implications of the microglia platform for stroke therapy.

The effect of extracorporeal shockwave on liposomal bupivacaine in a tibial plateau leveling osteotomy model.

OBJECTIVE: To determine the effect of extracorporeal shock wave (ESWT) on liposomal bupivacaine in a tibial-plateau-leveling osteotomy model. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Ten samples per group. METHODS: In addition to a control group (sham treatment), five treatment groups were defined as A, energy (E) 3 (0.22 mJ/mm2 ), 360 pulses per minute (p/m); B, E6 (0.29 mJ/mm2 ), 360 p/m; C, E8 (0.39 mJ/mm2 ), 360 p/m; D, E6, 480 p/m; E, E8 480 p/m. Two-milliliter aliquots of liposomal bupivacaine were placed in a gelatin chamber and treated with 1000 pulses according to group. All samples remained in the chamber for 170 seconds to reflect the longest treatment group. Free bupivacaine concentrations were determined after treatment with high-performance liquid chromatography. RESULTS: The median free bupivacaine concentration was reported as control, 1.90 mg/mL; A, 2.10 mg/mL; B, 2.03 mg/mL; C, 2.94 mg/mL; D, 2.71 mg/mL; E, 4.35 mg/mL. Groups C (P = .027), D (P = .034), and E (P = .002) were different from the control group. Groups C (P = .0025) and D (P = .0025) were different from group E. Additional intertreatment group differences were found. CONCLUSION: Extracorporeal shock wave therapy caused a dose-dependent release of bupivacaine; however, there was no significant release of bupivacaine from liposomes when ESWT was applied at currently recommended therapeutic settings in this model. CLINICAL SIGNIFICANCE: This in vitro study provides evidence that concurrent electrohydraulic ESWT and liposomal bupivacaine is likely safe at currently recommended settings, however, higher energy and pulse frequency settings should be avoided.

Light-induced liposomes for cancer therapeutics.

The emerging nanomedicine therapeutics have incorporated photonics technologies to develop precise medical treatment. Among the light regulated approaches, light-induced liposome technology has been explored and developed as a novel tool for spatiotemporal control of cargo release. Compared with the traditional liposome formulation, this triggering feature largely enhanced the therapeutic efficacy and minimized the side effects of the therapeutic substance. In this review paper, we discussed the basics of the light-induced liposomes including the engineering methods and photoresponsiveness mechanisms. We also reviewed current biomedical studies relating to light-induced liposome delivery systems, with an emphasis in the field of cancer therapy.

Multi-Model Investigation and Adaptive Estimation of the Acoustic Release of a Model Drug From Liposomes.

This paper researches a suitable mathematical model that can reliably predict the release of a model drug (namely calcein) from biologically targeted liposomal nanocarriers triggered by ultrasound. Using mathematical models, curve fitting is performed on a set of five experimental acoustic drug release runs from Albumin-, Estrone-, and RGD-based Drug Delivery Systems (DDS). The three moieties were chosen to target specific cancers using receptor-mediated endocytosis. The best-fitting mathematical model is then enhanced using a Kalman filtering (KF) algorithm to account for the statistics of the dynamic and measurements noise sequences in predicted drug release. Unbiased drug-release estimates are realized by implementing an online noise identification algorithm. The algorithm is first deployed in a simulated environment in which it was rigorously tested and compared with the correct solution. Then, the algorithm was used to process the five experimental datasets. The results suggest that the Adaptive Kalman Filter (AKF) is exceptionally good at handling drug release estimation problems with a priori unknown or with changing noise covariances. In comparison with the KF, the AKF approach exhibited as low as a 69% reduction in the level of error in estimating the drug release state. Finally, the proposed algorithm is not computationally demanding and is capable of online estimation tasks.

Rational Design of Photosynthesis-Inspired Nanomedicines.

The sun is the most abundant source of energy on earth. Phototrophs have discovered clever strategies to harvest this light energy and convert it to chemical energy for biomass production. This is achieved in light-harvesting complexes, or antennas, that funnel the exciton energy into the reaction centers. Antennas contain an array of chlorophylls, linear tetrapyrroles, and carotenoid pigments spatially controlled by neighboring proteins. This fine-tuned regulation of protein-pigment arrangements is crucial for survival in the conditions of both excess and extreme light deficit. Photomedicine and photodiagnosis have long been utilizing naturally derived and synthetic monomer dyes for imaging, photodynamic and photothermal therapy; however, the precise regulation of damage inflicted by these therapies requires more complex architectures. In this Account, we discuss how two mechanisms found in photosynthetic systems, photoprotection and light harvesting, have inspired scientists to create nanomedicines for more effective and precise phototherapies. Researchers have been recapitulating natural photoprotection mechanisms by utilizing carotenoids and other quencher molecules toward the design of photodynamic molecular beacons (PDT beacons) for disease-specific photoactivation. We highlight the seminal studies describing peptide-linked porphyrin-carotenoid PDT beacons, which are locally activated by a disease-specific enzyme. Examples of more advanced constructs include tumor-specific mRNA-activatable and polyionic cell-penetrating PDT beacons. An alternative approach toward harnessing photosynthetic processes for biomedical applications includes the design of various nanostructures. This Account will primarily focus on organic lipid-based micro- and nanoparticles. The phenomenon of nonphotochemical quenching, or excess energy release in the form of heat, has been widely explored in the context of porphyrin-containing nanomedicines. These quenched nanostructures can be implemented toward photoacoustic imaging and photothermal therapy. Upon nanostructure disruption, as a result of tissue accumulation and subsequent cell uptake, activatable fluorescence imaging and photodynamic therapy can be achieved. Alternatively, processes found in nature for light harvesting under dim conditions, such as in the deep sea, can be harnessed to maximize light absorption within the tissue. Specifically, high-ordered dye aggregation that results in a bathochromic shift and increased absorption has been exploited for the collection of more light with longer wavelengths, characterized by maximum tissue penetration. Overall, the profound understanding of photosynthetic systems combined with rapid development of nanotechnology has yielded a unique field of nature-inspired photomedicine, which holds promise toward more precise and effective phototherapies.

Near-infrared light triggered liposomes combining photodynamic and chemotherapy for synergistic breast tumor therapy.

Photodynamic therapy (PDT) as a promising noninvasive and effective treatment modality has been clinically approved for cancer therapy. However, the poor selectivity of tumor and hypoxia-induced resistance constrain PDT efficacy immensely. To further enhance PDT's potency, we developed a drug delivery system based on liposome combining PDT and chemotherapeutics. The lipophilic IR780 was loaded into the lipid bilayer while hydrophilic chemotherapeutic agent tirapazamine (TPZ) was encapsulated in the hydrophilic core. IR780 could generate reactive oxygen species and hypoxic microenvironment in local site because of the continuous consumption of oxygen, resulting in the TPZ encapsulated in the aqueous liposome chamber brings out TPZ radicals to cause DNA double-strand breaks and chromosome aberrations. In vivo studies demonstrated that the liposomes which encapsulate IR780 and TPZ showed great antitumor efficacy via combining photodynamic therapy with chemotherapy. Therefore, the investigation combines PDT and hypoxia-activated chemotherapy from the TPZ. It is a simple but effective liposome platform to achieve multiple synergistic antitumor efficacy and shows potential for clinical use.

PEG-coated vesicles from Pluronic/lipid mixtures for the carrying of photoactive erythrosine derivatives.

Liposomes are very attractive membrane models and excellent drug delivery systems. Concerning their drug delivery aspects, the mixing liposomes with biocompatible copolymers allows for stability and the incorporation of several drugs. We developed PEG coated vesicles from the mixture of DPPC and F127 Pluronic copolymer to obtain long-circulating nanoparticles (mixed vesicles). We employed an innovative process previously developed by us: a small amount of F127 mixed in DPPC, thin film preparation, followed by hydration (lipids plus F127) using a bath sonicator cleaner type, forming unilamellar spherical vesicles with diameter ∼100 nm. The formed PEG coated vesicles were incorporated with the xanthene dye Erythrosine B (ERY), and its ester derivatives as photosensitizers (PS) for photodynamic proposes. The F127/DPPC mixed vesicles promoted a higher PS incorporation, and with better thermal and kinetic stability, at least 60 days, when compared to conventional DPPC liposome. The binding constant and quenching analysis revealed that with a higher PS hydrophobicity, PS affinity increases toward the nanoparticle and results in a deeper PS location inside the lipid bilayer. An increment in the fluorescence quantum yield was observed, while the PS singlet oxygen generations remained high. Dialysis studies demonstrated that PS were released based on their hydrophobicity. Permeation analysis showed that all PS can reach the deeper regions of the skin. The Decyl Ester derivative/nanoparticle exhibited high photoactivity against Caco-2 cancer cells (in vitro studies). The PEG coated from F127/DPPC mixed vesicles are very promising nanocarriers for erythrosine and its derivatives.

Light-induced propulsion of a giant liposome driven by peptide nanofibre growth.

Light-driven nano/micromotors are attracting much attention, not only as molecular devices but also as components of bioinspired robots. In nature, several pathogens such as Listeria use actin polymerisation machinery for their propulsion. Despite the development of various motors, it remains challenging to mimic natural systems to create artificial motors propelled by fibre formation. Herein, we report the propulsion of giant liposomes driven by light-induced peptide nanofibre growth on their surface. Peptide-DNA conjugates connected by a photocleavage unit were asymmetrically introduced onto phase-separated giant liposomes. Ultraviolet (UV) light irradiation cleaved the conjugates and released peptide units, which self-assembled into nanofibres, driving the translational movement of the liposomes. The velocity of the liposomes reflected the rates of the photocleavage reaction and subsequent fibre formation of the peptide-DNA conjugates. These results showed that chemical design of the light-induced peptide nanofibre formation is a useful approach to fabricating bioinspired motors with controllable motility.

The effect of MLS laser radiation on cell lipid membrane.

INTRODUCTION: Authors of numerous publications have proved the therapeutic effect of laser irradiation on biological material, but the mechanisms at cellular and subcellular level are not yet well understood. OBJECTIVE: The aim of this study was to assess the effect of laser radiation emitted by the MLS M1 system (Multiwave Locked System) at two wavelengths (808 nm continuous and 905 nm pulsed) on the stability and fluidity of liposomes with a lipid composition similar to that of human erythrocyte membrane or made of phosphatidylocholine. MATERIAL AND METHODS: Liposomes were exposed to low-energy laser radiation at surface densities 195 mW/cm2 (frequency 1,000 Hz) and 230 mW/cm2 (frequency 2,000 Hz). Different doses of radiation energy in the range 0-15 J were applied. The surface energy density was within the range 0.46 - 4.9 J/cm 2. RESULTS: The fluidity and stability of liposomes subjected to such irradiation changed depending on the parameters of radiation used. CONCLUSIONS: Since MLS M1 laser radiation, depending on the parameters used, affects fluidity and stability of liposomes with the lipid content similar to erythrocyte membrane, it may also cause structural and functional changes in cell membranes.

Photo-triggered destabilization of nanoscopic vehicles by dihydroindolizine for enhanced anticancer drug delivery in cervical carcinoma.

The efficacy and toxicity of drugs depend not only on their potency but also on their ability to reach the target sites in preference to non-target sites. In this regards destabilization of delivery vehicles induced by light can be an effective strategy for enhancing drug delivery with spatial and temporal control. Herein we demonstrate that the photoinduced isomerization from closed (hydrophobic) to open isomeric form (hydrophilic) of a novel DHI encapsulated in liposome leads to potential light-controlled drug delivery vehicles. We have used steady state and picosecond resolved dynamics of a drug 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) incorporated in liposome to monitor the efficacy of destabilization of liposome in absence and presence UVA irradiation. Steady state and picosecond resolved polarization gated spectroscopy including the well-known strategy of solvation dynamics and Förster resonance energy transfer; reveal the possible mechanism out of various phenomena involved in destabilization of liposome. We have also investigated the therapeutic efficacy of doxorubicin (DOX) delivery from liposome to cervical cancer cell line HeLa. The FACS, confocal fluorescence microscopic and MTT assay studies reveal an enhanced cellular uptake of DOX leading to significant reduction in cell viability (∼40%) of HeLa followed by photoresponsive destabilization of liposome. Our studies successfully demonstrate that these DHI encapsulated liposomes have potential application as a smart photosensitive drug delivery system.

Dual-function nanostructured lipid carriers to deliver IR780 for breast cancer treatment: Anti-metastatic and photothermal anti-tumor therapy.

Cancer treatments that use a combination of approaches with the ability to affect multiple disease pathways have proven highly effective. The present study reports on CXCR4-targeted nanostructured lipid carriers (NLCs) with a CXCR4 antagonist AMD3100 in the shell (AMD-NLCs). AMD-NLCs loaded with IR780 (IR780-AMD-NLCs) reduced the invasiveness of cancer cells, while simultaneously mediating efficient tumor targeting and photothermal therapeutic outcomes. We present the combined effect of encapsulated IR780 on photothermal therapy and of the AMD3100 coating on tumor targeting, CXCR4 antagonism and inhibition of cancer cell invasion and breast cancer lung metastasis in vitro and in vivo. IR780-AMD-NLCs exhibited excellent IR780 loading capacity and AMD3100 coating efficiency. The photothermal properties of IR780 were improved by encapsulation in NLCs. The encapsulated IR780 displayed better heat generating efficiency than free IR780 when exposed to repeated laser irradiation. CXCR4 antagonism and cell invasion assays confirmed that IR780-AMD-NLCs fully inhibited CXCR4 while IR780-NLCs did not function as CXCR4 antagonists. AMD3100-coated NLCs accumulated at high levels in tumors, as judged by in vivo imaging and biodistribution assays. Furthermore, CXCR4-targeted NLCs exhibited an encouraging photothermal anti-tumor effect as well as anti-metastatic efficacy in vivo. These findings suggest that this simple and stable CXCR4-targeted IR780 delivery system holds great promise for prevention of metastasis and for photothermal treatment of tumors. STATEMENT OF SIGNIFICANCE: Breast cancer is a major threat to human health, it is not the primary breast tumor that is ultimately responsible for the majority of deaths, but the tumor metastasis, which frequently follows a specific pattern of dissemination. We report development of a novel dual-function nanostructured lipid carrier (NLC) for breast cancer treatment. The carrier encapsulates NIR dye IR780 in its core and contains antagonist of the chemokine receptor CXCR4 in its shell. Our results show that by combining the CXCR4 antagonism with photothermal effect of the dye leads to remarkable antitumor and antimetastatic activity in syngeneic orthotopic model of metastatic breast cancer. Furthermore, the developed system also shows a theranostic potential due to NIR fluorescence of the encapsulated dye.

Ultrasensitive Phototriggered Local Anesthesia.

An injectable local anesthetic producing repeatable on-demand nerve block would be desirable for pain management. Here we present a phototriggerable device to achieve repeatable and adjustable on-demand local anesthesia in superficial or deep tissues, consisting of gold nanorods attached to low temperature sensitive liposomes (LTSL). The particles were loaded with tetrodotoxin and dexmedetomidine. Near-infrared light (NIR, 808 nm, continuous wave) could heat gold nanorods at low fluence (short duration and low irradiance), leading to rapid release of payload. In vivo, 1-2 min of irradiation at ≤272 mW/cm2 produced repeatable and adjustable on-demand infiltration anesthesia or sciatic nerve blockade with minimal toxicity. The nerve block intensity and duration correlated with the irradiance and duration of the applied light.

Loss of gas from echogenic liposomes exposed to pulsed ultrasound.

The destruction of echogenic liposomes (ELIP) in response to pulsed ultrasound excitations has been studied acoustically previously. However, the mechanism underlying the loss of echogenicity due to cavitation nucleated by ELIP has not been fully clarified. In this study, an ultra-high speed imaging approach was employed to observe the destruction phenomena of single ELIP exposed to ultrasound bursts at a center frequency of 6 MHz. We observed a rapid size reduction during the ultrasound excitation in 139 out of 397 (35%) ultra- high-speed recordings. The shell dilation rate, which is defined as the microbubble wall velocity divided by the instantaneous radius, [Formula: see text] /R, was extracted from the radius versus time response of each ELIP, and was found to be correlated with the deflation. Fragmentation and surface mode vibrations were also observed and are shown to depend on the applied acoustic pressure and initial radius. Results from this study can be utilized to optimize the theranostic application of ELIP, e.g. by tuning the size distribution or the excitation frequency.

Preclinical Evaluation of Promitil, a Radiation-Responsive Liposomal Formulation of Mitomycin C Prodrug, in Chemoradiotherapy.

PURPOSE: To examine the effect of radiation on in vitro drug activation and release of Promitil, a pegylated liposomal formulation of a mitomycin C (MMC) lipid-based prodrug; and examine the efficacy and toxicity of Promitil with concurrent radiation in colorectal cancer models. METHODS AND MATERIALS: Promitil was obtained from Lipomedix Pharmaceuticals (Jerusalem, Israel). We tested the effects of radiation on release of active MMC from Promitil in vitro. We next examined the radiosensitization effect of Promitil in vitro. We further evaluated the toxicity of a single injection of free MMC or Promitil when combined with radiation by assessing the effects on blood counts and in-field skin and hair toxicity. Finally, we compared the efficacy of MMC and Promitil in chemoradiotherapy using mouse xenograft models. RESULTS: Mitomycin C was activated and released from Promitil in a controlled-release profile, and the rate of release was significantly increased in medium from previously irradiated cells. Both Promitil and MMC potently radiosensitized HT-29 cells in vitro. Toxicity of MMC (8.4 mg/kg) was substantially greater than with equivalent doses of Promitil (30 mg/kg). Mice treated with human-equivalent doses of MMC (3.3 mg/kg) experienced comparable levels of toxicity as Promitil-treated mice at 30 mg/kg. Promitil improved the antitumor efficacy of 5-fluorouracil-based chemoradiotherapy in mouse xenograft models of colorectal cancer, while equitoxic doses of MMC did not. CONCLUSIONS: We demonstrated that Promitil is an attractive agent for chemoradiotherapy because it demonstrates a radiation-triggered release of active drug. We further demonstrated that Promitil is a well-tolerated and potent radiosensitizer at doses not achievable with free MMC. These results support clinical investigations using Promitil in chemoradiotherapy.

Investigation of Particle Accumulation, Chemosensitivity and Thermosensitivity for Effective Solid Tumor Therapy Using Thermosensitive Liposomes and Hyperthermia.

Doxorubicin (Dox) loaded thermosensitive liposomes (TSLs) have shown promising results for hyperthermia-induced local drug delivery to solid tumors. Typically, the tumor is heated to hyperthermic temperatures (41-42 °C), which induced intravascular drug release from TSLs within the tumor tissue leading to high local drug concentrations (1-step delivery protocol). Next to providing a trigger for drug release, hyperthermia (HT) has been shown to be cytotoxic to tumor tissue, to enhance chemosensitivity and to increase particle extravasation from the vasculature into the tumor interstitial space. The latter can be exploited for a 2-step delivery protocol, where HT is applied prior to i.v. TSL injection to enhance tumor uptake, and after 4 hours waiting time for a second time to induce drug release. In this study, we compare the 1- and 2-step delivery protocols and investigate which factors are of importance for a therapeutic response. In murine B16 melanoma and BFS-1 sarcoma cell lines, HT induced an enhanced Dox uptake in 2D and 3D models, resulting in enhanced chemosensitivity. In vivo, therapeutic efficacy studies were performed for both tumor models, showing a therapeutic response for only the 1-step delivery protocol. SPECT/CT imaging allowed quantification of the liposomal accumulation in both tumor models at physiological temperatures and after a HT treatment. A simple two compartment model was used to derive respective rates for liposomal uptake, washout and retention, showing that the B16 model has a twofold higher liposomal uptake compared to the BFS-1 tumor. HT increases uptake and retention of liposomes in both tumors models by the same factor of 1.66 maintaining the absolute differences between the two models. Histology showed that HT induced apoptosis, blood vessel integrity and interstitial structures are important factors for TSL accumulation in the investigated tumor types. However, modeling data indicated that the intraliposomal Dox fraction did not reach therapeutic relevant concentrations in the tumor tissue in a 2-step delivery protocol due to the leaking of the drug from its liposomal carrier providing an explanation for the observed lack of efficacy.

In Vitro and In Vivo Evaluation of Targeted Sunitinib-Loaded Polymer Microbubbles Against Proliferation of Renal Cell Carcinoma.

OBJECTIVES: The poor safety profile of sunitinib capsules has encouraged the identification of targeted drug delivery systems against renal cell carcinoma. This study aimed to explore the effect of sunitinib-loaded microbubbles along with ultrasound (US) treatment on proliferation and apoptosis of human GRC-1 granulocyte renal carcinoma cells in vitro and in vivo (xenograft tumor growth in nude mice). METHODS: Liposomes containing sunitinib were prepared by using the transmembrane ammonium sulfate gradient method and then absorbed into polymer microbubbles to generate sunitinib-loaded microbubbles. Entrapment of sunitinib was verified by 25-25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol staining. GRC-1 cells were treated with microbubbles alone, liposomes alone, sunitinib alone, sunitinib-loaded microbubbles without and with US, and no treatment (control). Cell survival and apoptosis were assessed at 12, 24, and 48 hours after treatment. Xenograft tumors were induced by implantation of GRC-1 cells in nude mice. The animals with tumors were then randomly assigned to sunitinib alone, sunitinib-loaded microbubbles - US, sunitinib-loaded microbubbles + US, and no treatment (control; n = 10 per group). The tumor volumes were analyzed on the 7th, 15th, and 21st days. RESULTS: The sunitinib entrapment efficiency in the liposomes was approximately 78%. The effective sunitinib concentration in each group was 0.1 µg/mL. The sunitinib-loaded microbubble + US group showed a lower in vitro cell survival rate (P < .001) compared with the other groups. Greater in vivo inhibition of xenograft tumor growth was also observed in the sunitinib-loaded microbubble + US group compared with the other groups. CONCLUSIONS: Combined sunitinib-loaded microbubbles and US treatment significantly inhibits growth of renal carcinoma cells both in vitro and in vivo.

A Disposable Microfluidic Device for Controlled Drug Release from Thermal-Sensitive Liposomes by High Intensity Focused Ultrasound.

The drug release triggered thermally by high intensity focused ultrasound (HIFU) has been considered a promising drug delivery strategy due to its localized energy and non-invasive characters. However, the mechanism underlying the HIFU-mediated drug delivery remains unclear due to its complexity at the cellular level. In this paper, micro-HIFU (MHIFU) generated by a microfluidic device is introduced which is able to control the drug release from temperature-sensitive liposomes (TSL) and evaluate the thermal and mechanical effects of ultrasound on the cellular drug uptake and apoptosis. By simply adjusting the input electrical signal to the device, the temperature of sample can be maintained at 37 °C, 42 °C and 50 °C with the deviation of ± 0.3 °C as desired. The flow cytometry results show that the drug delivery under MHIFU sonication leads to a significant increase in apoptosis compared to the drug release by incubation alone at elevated temperature of 42 °C. Furthermore, increased squamous and protruding structures on the surface membrane of cells were detected by atomic force microscopy (AFM) after MHIFU irradiation of TSL. We demonstrate that compared to the routine HIFU treatment, MHIFU enables monitoring of in situ interactions between the ultrasound and cell in real time. Furthermore, it can quantitatively analyze and characterize the alterations of the cell membrane as a function of the treatment time.