Biodegradable Polymersomes with Structure Inherent Fluorescence and Targeting Capacity for Enhanced Photo-Dynamic Therapy.

Biodegradable nanostructures displaying aggregation-induced emission (AIE) are desirable from a biomedical point of view, due to the advantageous features of loading capacity, emission brightness, and fluorescence stability. Herein, biodegradable polymers comprising poly (ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-P(CLgTMC)), with tetraphenylethylene pyridinium-TMC (PAIE) side chains have been developed, which self-assembled into well-defined polymersomes. The resultant AIEgenic polymersomes are intrinsically fluorescent delivery vehicles. The presence of the pyridinium moiety endows the polymersomes with mitochondrial targeting ability, which improves the efficiency of co-encapsulated photosensitizers and improves therapeutic index against cancer cells both in vitro and in vivo. This contribution showcases the ability to engineer AIEgenic polymersomes with structure inherent fluorescence and targeting capacity for enhanced photodynamic therapy.

Conducting Polyetheretherketone Nanocomposites with an Electrophoretically Deposited Bioactive Coating for Bone Tissue Regeneration and Multimodal Therapeutic Applications.

The use of polyetheretherketone (PEEK) has grown exponentially in the biomedical field in recent decades because of its outstanding biomechanical properties. However, its lack of bioactivity/osteointegration remains an unresolved issue toward its wide use in orthopedic applications. In this work, graphene nanosheets have been incorporated into PEEK to obtain multifunctional nanocomposites. Because of the formation of an electrical percolation network and the π-π* conjugation between graphene and PEEK, the resulting composites have achieved 12 orders of magnitude enhancement in their electrical conductivity and thereby enabled electrophoretic deposition of a bioactive/antibacterial coating consisting of stearyltrimethylammonium chloride-modified hydroxyapatite. The coated composite implant shows significant boosting of bone marrow mesenchymal stem cell proliferation in vitro. In addition, the strong photothermal conversion effect of the graphene nanofillers has enabled laser-induced heating of our nanocomposite implants, where the temperature of the implant can reach 45 °C in 150 s. The unique multifunctionality of the implant has also been demonstrated for photothermal applications such as enhancing bacterial eradication and tumor cell inhibition, as well as bone tissue regeneration in vivo. The results suggest the strong potential of our multifunctional implant in bone repair applications as well as multimodal therapy of challenging bone diseases such as osteosarcoma and osteomyelitis.

Photothermally Active Cryogel Devices for Effective Release of Antimicrobial Peptides: On-Demand Treatment of Infections.

There has been significant interest in the use of peptides as antimicrobial agents, and peptide containing hydrogels have been proposed as biological scaffolds for various applications. Limited stability and rapid clearance of small molecular weight peptides pose challenges to their widespread implementation. As a common approach, antibacterial peptides are physically loaded into hydrogel scaffolds, which leads to continuous release through the passive mode with spatial control but provides limited control over drug dosage. Although utilization of peptide covalent linkage onto hydrogels addresses partially this problem, the peptide release is commonly too slow. To alleviate these challenges, in this work, maleimide-modified antimicrobial peptides are covalently conjugated onto furan-based cryogel (CG) scaffolds via the Diels-Alder cycloaddition at room temperature. The furan group offers a handle for specific loading of the peptides, thus minimizing passive and burst drug release. The porous nature of the CG matrix provides rapid loading and release of therapeutic peptides, apart from high water uptake. Interfacing the peptide adduct containing a CG matrix with a reduced graphene oxide-modified Kapton substrate allows "on-demand" photothermal heating upon near-infrared (NIR) irradiation. A fabricated photothermal device enables tunable and efficient peptide release through NIR exposure to kill bacteria. Apart from spatial confinement offered by this CG-based bandage, the selective ablation of planktonic Staphylococcus aureus is demonstrated. It can be envisioned that this modular "on-demand" peptide-releasing device can be also employed for other topical applications by appropriate choice of therapeutic peptides.

Shape-transformation of polymersomes from glassy and crosslinkable ABA triblock copolymers.

Recent developments in the field of polymer vesicles, i.e. polymersomes, have demonstrated that disrupting the equilibrium conditions of the milieu could lead to shape transformation into stable non-spherical morphologies, bringing on-demand shape control to reality and bearing great promise for cell mimicry and a variety of biomedical applications. Here, we studied the self-assembly behavior of glassy amphiphilic triblock copolymers, poly(ethylene glycol)-block-polystyrene-stat-poly(coumarin methacrylate)-block-poly(ethylene glycol) (PEG-b-P(S-stat-CMA)-b-PEG), and their response to various stimuli. By changing the respective molecular weights of both the hydrophobic P(S-stat-CMA) and the hydrophilic PEG blocks, we varied the hydrophobic volume fraction thereby accessing a range of morphologies from spherical and worm-like micelles, as well as polymersomes. For the latter, we observed that slow osmotic pressure changes induced by dialysis led to a decrease in size while rapid osmotic pressure changes by addition of a PEG fusogen led to morphological transformations into rod-like and tubular polymersomes. We also found out that chemically crosslinking the vesicles before inducing osmotic pressure changes led to the vesicles exhibiting hypotonic shock, atypical for glassy polymersomes. We believe that this approach combining the robustness of triblock copolymers and light-based transformations will help expand the toolbox to design ever more complex biomimetic constructs.

Facile fabrication of shell crosslinked microcapsule by visible light induced graft polymerization for enzyme encapsulation.

Immobilization of enzymes can effectively improve their stability, facilitate their recycling and reduce the cost, which is of great significance for the development of highly efficient biocatalysis technology. Here a simple strategy to encapsulate enzymes into polymeric microcapsules fabricated by visible light induced graft polymerization on a removable template was developed. The strategy showed a high degree of enzyme loading and excellent reusability of the immobilized enzyme.

Molecular environment and reactivity in gels and colloidal solutions under identical conditions.

A PEG-Tyr block copolymer forms a kinetically stable colloidal solution in water at room temperature which undergoes an irreversible conversion to a gel phase upon heating. A micellar solution and a gel can therefore be studied under identical experimental conditions. This made it possible to compare physical properties and chemical reactivity of micelles and gels in identical chemical environments and under identical conditions. EPR spectra of the spin-labelled copolymer showed that tyrosine mobility in gels was slightly reduced compared to micelles. Chemical reactivity was studied using photochemical degradation of tyrosine and tyrosine dimerization, in the absence and in the presence of an Fe(iii) salt. The reactivity trends were explained by reduced tyrosine mobility in the gel environment. The largest reactivity difference in gels and micelles was observed for bimolecular dityrosine formation which was also attributed to the reduction in molecular mobility.

Rapid Buildup Arrays with Orthogonal Biochemistry Gradients via Light-Induced Thiol-Ene "Click" Chemistry for High-Throughput Screening of Peptide Combinations.

The concept of high-throughput screening sheds new light on fabrication and analysis of materials. Herein, a combinatorial surface-modified platform with biochemical gradients was developed through thiol-ene "click" chemistry by adjusting the intensity of ultraviolet (UV) irradiation. Contact angle, X-ray photoelectron spectroscopy, and ellipsometry measurement results demonstrated that the sulfhydryl molecules including polyethylene glycol and RGD (arginine-glycine-aspartic acid) and REDV (arginine-glutamic acid-aspartic acid-valine) peptides can be directly attached onto alkene-modified substrates, in which the graft density can be well controlled by the intensity of UV irradiation. The multistep attachment of different molecules onto substrates is archived via the multistep UV-initiated thiol-ene "click" reaction. The high-throughput arrays with the gradient density of single ligand and the orthogonal gradient density of two ligands were rapidly fabricated via the one-step UV gradient irradiation and the two-step orthogonal UV gradient-initiated thiol-ene "click" reaction. Endothelial cells (ECs) and smooth muscle cells (SMCs) were cocultured on the array with the orthogonal gradient density of RGD and REDV to screen the peptide combination with high EC selectivity, which is essential for in situ endothelialization during stent implant. From 64, 8 × 8, combinations investigated, a special combinatorial surface representing the really high competitiveness of ECs over SMCs was screened. This platform puts forward a facile, high-throughput method to study the combinatorial variation of biochemical signals to cell behavior.

Cell encapsulation spatially alters crosslink density of poly(ethylene glycol) hydrogels formed from free-radical polymerizations.

Photopolymerizable poly(ethylene glycol) (PEG) hydrogels are a promising platform for chondrocyte encapsulation and cartilage tissue engineering. This study demonstrates that during the process of encapsulation, chondrocytes alter the formation of PEG hydrogels leading to a reduction in the bulk and local hydrogel crosslink density. Freshly isolated chondrocytes were shown to interact with hydrogel precursors, in part through thiol-mediated events between dithiol crosslinkers and cell surface free thiols, depleting crosslinker concentration and causing a reduction in the bulk hydrogel crosslink density. This effect was more pronounced with increasing cell density at the time of encapsulation. Encapsulation of chondrocytes in fluorescently labeled hydrogels exhibited a gradient in hydrogel density around the cell, which was abrogated by treatment of the cells with the antioxidant estradiol prior to encapsulation. This gradient led to spatial variations in the degradation behavior of a hydrolytically degradable PEG hydrogel, creating regions devoid of hydrogel surrounding cells. Collectively, findings from this study indicate that the antioxidant defense mechanisms in chondrocytes alter the resultant properties of PEG hydrogels formed by free-radical polymerizations. These interactions will have a significant impact on tissue engineering, affecting the local microenvironment around cells and how tissue grows within the hydrogels. STATEMENT OF SIGNIFICANCE: Cell encapsulations in synthetic hydrogels formed by free-radical polymerizations offer numerous benefits for tissue engineering. Herein, we studied cartilage cells and identified that during encapsulation, cells interfered with hydrogel formation through two distinct mechanisms. Thiol-mediated events between monomers led to monomer depletion and a lower crosslinked hydrogel. Cells' antioxidant defense mechanisms interfered with free-radicals and inhibited hydrogel formation near the cell. These cell-mediated effects led to softer hydrogels and created unique hydrogel degradations patterns causing rapid degradation around the cells. The latter has benefits for tissue engineering, where these regions provide space for tissue growth. Overall, this study demonstrates that cells play a key role in how the hydrogel structure forms when cells are present.

Photo-crosslinked anhydride-modified polyester and -ethers for pH-sensitive drug release.

Photo-crosslinkable polymers have a great potential for the delivery of sensitive drugs. They allow preparation of drug releasing devices by photo-crosslinking, thus avoiding high processing temperatures. In this study, the hydrolysis behavior and drug release of three different photo-crosslinkable poly(ether anhydride)s and one poly(ester anhydride) were investigated. Three-arm poly(ethylene glycol) or polycaprolactone was reacted with succinic anhydride to obtain carboxylated macromers, and further functionalized with methacrylic anhydride to form methacrylated marcromers with anhydride linkages. The synthetized macromers were used to prepare photo-crosslinked matrices with different hydrolytic degradation times for active agent release purposes. The hydrolysis was clearly pH-sensitive: polymer networks degraded slowly in acidic conditions, and degradation rate increased as the pH shifted towards basic conditions. Drug release was studied with two water-soluble model drugs lidocaine (234 mol/g) and vitamin B12 (1355 g/mol). Vitamin B12 was released mainly due to polymer network degradation, whereas smaller molecule lidocaine was released also through diffusion and swelling of polymer network. Only a small amount of vitamin B12 was released in acidic conditions (pH 1.3 and pH 2.1). These polymers have potential in colon targeted drug delivery as the polymer could protect sensitive drugs from acidic conditions in the stomach, and the drug would be released as the conditions change closer to neutral pH in the intestine.

Resistive Water Sensors Based on PEDOT:PSS-g-PEGME Copolymer and Laser Treatment for Water Ingress Monitoring Systems.

Water sensors are a type of level sensor that can be used in various applications requiring the sensing of water levels, such as in dams, nuclear power plants, water pipes, water tanks, and dehumidifiers. In particular, water sensors in water ingress monitoring systems (WIMS) protect lives and property from disasters caused by water leakage and flooding. Here, a resistive water sensor for WIMS that incorporates poly(3,4-ethylenedioxythinophene):poly(styrene sulfonate) (PEDOT:PSS) grafted with poly(ethylene glycol) methyl ether (PEGME) (PEDOT:PSS-g-PEGME copolymer) as high-conductivity electrodes and laser-treated PEDOT:PSS-g-PEGME copolymer as the low-conductivity resistive component is reported. The configuration of the water sensor is modeled as two parallel resistors (Rlaser treated PEDOT:PSS||Rwater) when water comes into contact with the sensor surface. The two-resistor configuration exhibits a better performance in comparison with single-resistor configurations comprising only PEDOT:PSS-g-PEGME copolymer or laser-treated PEDOT:PSS-g-PEMGE copolymer. Moreover, PEDOT:PSS-g-PEGME copolymer is applied to the sensor to improve the stability of PEDOT:PSS in water. We demonstrate that the sensor can detect the water level in real time with high sensitivity and accuracy, and thus has potential in applications for monitoring water-related hazards.

Photoinduced chitosan-PEG hydrogels with long-term antibacterial properties.

Photochemical processes offer the possibility of preparing functional hydrogels under green conditions that are compatible with both synthetic and natural polymers. In this study, chitosan-based poly(ethylene) glycol (PEG) were successfully synthesized under light irradiation in aqueous medium. Kinetic studies under irradiation showed that less than 2 min were necessary to obtain fully cross-linked networks. Thermomechanical analyses and swelling experiments indicated that introduction of chitosan overall weakens the hydrogel network but can create domains of higher thermal stability than the PEG-alone structure. The resulting chitosan-PEG hydrogels demonstrated a tremendous inhibition (100%) of bacterial growth (Escherichia coli and Staphylococcus aureus). After 6 months' ageing, one of the hydrogels preserved a high antifouling activity against Escherichia coli. This interesting result, which could be correlated with the network features, demonstrates the strong potential of these photochemical methods to obtain robust bio-functional materials.

A shear-thinning adhesive hydrogel reinforced by photo-initiated crosslinking as a fit-to-shape tissue sealant.

Surgical sealants suitable for wounds with non-flat complex geometries are still a challenge to fulfill clinical requirements. Herein, a novel fit-to-shape sealant enhanced by photo-initiated crosslinking was developed utilizing maleic anhydride modified chitosan (MCS), benzaldehyde-terminated PEG (PEGDF) and polyethylene glycol diacrylate (PEGDA). Initially, the shear-thinning hydrogel prepared through the Schiff-base linkage between MCS and PEGDF could be injected into target sites, remolded to conform to a wound with non-flat complex geometry, and remain on the wound, avoiding adverse liquid leakage. Under illumination with ultra-violet (UV) light, the hydrogel was solidified in situ rapidly to adopt the wound contour and enhanced in adhesive strength to seal defects of the tissue. In addition, the hydrogel exhibits stability in extreme pH environments (pH = 1) and has potential to treat wounds inside the stomach with the existence of gastric acid. Moreover, the hydrogel can be applied as adhesive wound dressings through in situ 3D printing. Taken together, the fit-to-shape sealant enhanced by photo-initiated crosslinking can be considered as promising tissue adhesives for wound closure and other biomedical applications.

PEGylated doxorubicin cloaked nano-graphene oxide for dual-responsive photochemical therapy.

Graphene oxide (GO) owns huge surface area and high drug loading capacity for aromatic molecules, such as doxorubicin (DOX). However, its biocompatibility is poor and it might agglomerate in physiological conditions. Chemical modification of GO with hydrophilicpolymer, especially PEGylation, was a common method to improve its biocompatibility. But the chemical modification of GO was complicated, and its drug loading capacity might be reduced because of the occupation of its functional groups. In this study, DOX-PEG polymers with different PEG molecular weights were synthesized to modify nano graphene oxide (NGO) to simultaneously realize the solubilization of NGO and the high loading capacity of DOX. The result showed that the drug release of NGO@DOX-PEG was pH sensitive. NIR irradiation could augment the drug release, cellular uptake, cytotoxicity and nuclear translocation of nanodrugs. Among the three kinds of nanodrugs, NGO@DOX-PEG5K was superior to others. It suggested that after conjugating with PEG, the bond between DOX-PEG and NGO was weakened, which resulted in a better drug release and treatment effect. In summary, the NIR and pH dual-responsive NGO@DOX-PEG nanodrugs were developed by noncovalent modification, and it demonstrated excellent biocompatibility and photochemical therapeutic effect, presenting a promising candidate for antitumor therapy, especially NGO@DOX-PEG5K.

Enhanced osteogenic activity of phosphorylated polyetheretherketone via surface-initiated grafting polymerization of vinylphosphonic acid.

Polyetheretherketone (PEEK) is considered to be a prime candidate with the potential to replace biomedical metallic materials as an orthopedic and dental implant on account of its elastic modulus similar to that of human cortical bone. Unfortunately, its biomedical application is impeded by the bioinert surface property and inferior osteogenic activity. In this work, phosphate groups were incorporated onto the PEEK surface through a single-step UV-initiated graft polymerization of vinylphosphonic acid. Diffuse reflectance Fourier transform infrared spectroscopy (DRFTIR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) revealed that phosphate groups were successfully introduced onto the PEEK surface without apparently altering its surface topographical feature and roughness. Water contact angle measurements diclosed the increasing hydrophilia after surface phosphonation. In vitro cell adhesion, spreading, proliferation, alkaline phosphatase activity, extracellular matrix mineralization, and real-time PCR analyses showed enhanced adhesion, spreading, proliferation and osteogenic differentiation of MC3T3-E1 osteoblast on the surface-phosphorylated PEEK. An in vivo biological evaluation in the rabbit tibiae proximal defect model by means of a histological analysis confirmed that the surface-phosphorylated PEEK had improved bone-implant contact. The obtained results indicate that enhanced osteogenic activity to surface-phosphorylated PEEK, which gives positive information of its potential applications in orthopedic and dental implants.

A one-step ultrasonic irradiation assisted strategy for the preparation of polymer-functionalized carbon quantum dots and their biological imaging.

Fluorescent carbon nanoparticles (FCNs) have gradually become the most promising alternative candidates to other traditional fluorescent nanomaterials for biological applications on account of their excellent fluorescence property and remarkable biocompatibility. Although many methods have reported on the preparation of FCNs, to date, no studies have reported the preparation of polymers of functionalized FCNs. A high-efficiency method was developed in this work to synthesize high-quality poly(ethylene oxide) (PEG)-functionalized FCNs from cigarette ash and thiol group-containing PEG via a facile one-pot ultrasonic irradiation treatment. A series of characterization techniques demonstrated the uniform nanoscale size, good fluorescence stability, high water dispersibility and remarkable biocompatibility of the generated FCNs. Furthermore, cell imaging was easily achieved at high resolution using the synthetic FCNs as probes, which validates their potential for bioimaging applications. In summary, an efficient one-pot strategy is reported for the first time on the preparation of PEG-functionalized FCNs with the assistance of ultrasonic irradiation. This method should be of great research interest for the fabrication of other polymer-functionalized FCNs with designable properties and functions.

Linear energy transfer (LET) spectra and survival fraction distribution based on the CR-39 plastic charged-particle detector in a spread-out Bragg peak irradiation by a 12C beam.

Facilities for heavy ion therapies are steadily increasing in number worldwide. One of the advantages of heavy ions is their high relative biological effect (RBE). In a model used at NIRS (National Institute of Radiological Sciences), linear energy transfer (LET) spectra are required to estimate biological dose (physical dose × RBE). The CR-39 plastic charged-particle detector (CR-39) is suitable for measurement of LET. For the present study, done at the Gunma University Heavy Ion Medical Center (GHMC), we measured LET spectra at 11 depths in spread-out Bragg peak (SOBP) irradiation by a 12C beam of 380 MeV/u. The lower threshold of the CR-39 to measure LET was about 5 keV µm-1 due to poor sensitivity for low LET. Then we calculated biological dose and survival fraction distributions and compared them with treatment planning results at GHMC. We used Monte Carlo simulation (Geant4) to calculate LET spectra. The simulation results were in good agreement with the experimental spectra. Moreover, the biological dose and survival fraction distributions estimated from the CR-39 reproduced the treatment planning. The CR-39 is suitable for estimating biological dose in carbon ion therapy.

Longevity of bond strength of resin cements to root dentine after radiation therapy.

AIM: To evaluate the bond strength and adhesive interface between several resin cements and root dentine immediately and 6 months after radiotherapy. METHODOLOGY: Sixty maxillary canines were selected and randomly assigned to two groups (n = 30): one group was not irradiated and the other one was subjected to a cumulative radiation dose of 60 Gy. The teeth were sectioned to obtain roots 16 mm long and the canals were prepared with the Reciproc system (R50) and filled using a lateral condensation technique with an epoxy resin sealer. Each group was divided into three subgroups (n = 10) according to the resin cement used for fibreglass fibre post cementation: RelyX-U200, Panavia-F2.0 and RelyX ARC. The posts were cemented in accordance with the manufacturer's recommendations. Three 1-mm-thick dentine slices were then obtained from each root third. The first two slices in the crown-apex direction of each third were selected for the push-out test. The failure mode after debonding was determined with a stereo microscope. The third slice from each root third was selected for scanning electron microscopy (SEM) analyses to examine the resin cement-dentine interface with 100, 1000, 2000 and 4000× magnification. Bond strength data were analysed by anova and Tukey's test (α = 0.05). RESULTS: Significantly lower bond strength (P < 0.0001) was obtained after irradiation compared to nonirradiated teeth. RelyX-U200 cemented fibre posts had the higher bond strength (15.17 ± 5.89) compared with RelyX ARC (P < 0.001) and Panavia-F2.0 (P < 0.001). The evaluation after 6 months revealed lower bond strength values compared to the immediate values (P < 0.001) for irradiated and nonirradiated teeth. Cohesive failures occurred in the irradiated dentine. SEM revealed fractures, microfractures and fewer collagen fibres in irradiated root dentine. RelyX-U200 and Panavia-F2.0 were associated with a juxtaposed interface of the cement with the radicular dentine in irradiated and nonirradiated teeth, and for RelyX ARC, hybrid layer formation and tags were observed in both irradiated and nonirradiated teeth. CONCLUSION: Radiation was associated with a decrease in the push-out bond strength and with lower resin cement/root dentine interface adaptation. Self-adhesive resin cement was a better alternative for fibre post cementation in teeth subjected to radiation therapy. The bond strength decreased after 6 months.

Decorating an individual living cell with a shell of controllable thickness by cytocompatible surface initiated graft polymerization.

Surface engineering of individual living cells is a promising field for cell-based applications. However, engineering individual cells with controllable thickness by chemical methods has been rarely studied. This article describes the development of a new cytocompatible chemical strategy to decorate individual living cells. The thicknesses of the crosslinked shells could be conveniently controlled by the irradiation time, visible light intensity, or monomer concentration. Moreover, the lag phase of the yeast cell division was extended and their stability against lysis was improved, which could also be tuned by controlling the shell thickness.

IR-780-loaded polymeric micelles enhance the efficacy of photothermal therapy in treating breast cancer lymphatic metastasis in mice.

Cancer metastasis is responsible for over 90% of breast cancer-related deaths, and inhibiting lymph node metastasis is an option to treat metastatic disease. Herein, we report the use of IR-780-loaded polymeric micelles (IPMs) for effective photothermal therapy (PTT) of breast cancer lymphatic metastasis. The IPMs were nanometer-sized micelles with a mean diameter of 25.6 nm and had good stability in simulated physiological solutions. Under 808-nm laser irradiation, IPMs exhibited high heat-generating capability in both in vitro and in vivo experiments. After intravenous injection, IPMs specifically accumulated in the tumor and metastatic lymph nodes and penetrated into these tissues. Moreover, a single IPMs treatment plus laser irradiation significantly inhibited primary tumor growth and suppressed lymphatic metastasis by 88.2%. Therefore, IPMs are an encouraging platform for PTT applications in treatment of metastatic breast cancer.

Focused Ultrasound Hyperthermia Mediated Drug Delivery Using Thermosensitive Liposomes and Visualized With in vivo Two-Photon Microscopy.

The future of nanomedicines in oncology requires leveraging more than just the passive drug accumulation in tumors through the enhanced permeability and retention effect. Promising results combining mild hyperthermia (HT) with lyso-thermosensitive liposomal doxorubicin (LTSL-DOX) has led to improved drug delivery and potent antitumor effects in pre-clinical studies. The ultimate patient benefit from these treatments can only be realized when robust methods of HT can be achieved clinically. One of the most promising methods of non-invasive HT is the use of focused ultrasound (FUS) with MRI thermometry for anatomical targeting and feedback. MRI-guided focused ultrasound (MRgFUS) is limited by respiratory motion and large blood vessel cooling. In order to translate exciting pre-clinical results to the clinic, novel heating approaches capable of overcoming the limitations on clinical MRgFUS+HT must be tested and evaluated on their ability to locally release drug from LTSL-DOX. Methods: In this work, a new system is described to integrate focused ultrasound (FUS) into a two-photon microscopy (2PM) setting to image the release of drug from LTSL-DOX in real-time during FUS+HT in vivo. A candidate scheme for overcoming the limitations of respiratory motion and large blood vessel cooling during MRgFUS+HT involves applying FUS+HT to 42°C in short ~30s bursts. The spatiotemporal drug release pattern from LTSL-DOX as a result is quantified using 2PM and compared against continuous (3.5min and 20min at 42°C) FUS+HT schemes and unheated controls. Results: It was observed for the first time in vivo that these short duration temperature elevations could produce substantial drug release from LTSL-DOX. Ten 30s bursts of FUS+HT was able to achieve almost half of the interstitial drug concentration as 20min of continuous FUS+HT. There was no significant difference between the intravascular area under the concentration-time curve for ten 30s bursts of FUS+HT and 3.5min of continuous FUS+HT. Conclusion: We have successfully combined 2PM with FUS+HT for imaging the release of DOX from LTSL-DOX in vivo in real-time, which will permit the investigation of FUS+HT heating schemes to improve drug delivery from LTSL-DOX. We have evaluated the ability to release DOX in short 30s FUS+HT bursts to 42°C as a method to overcome limitations on clinical MRgFUS+HT and have found that such exposures are capable of releasing measurable amounts of drug. Such an exposure has the potential to overcome limitations that hamper conventional MRgFUS+HT treatments in targets that are associated with substantial tissue motion.