Luis Solorio

Gelatin microspheres crosslinked with genipin for local delivery of growth factors

A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2–6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2‐loaded microspheres caused a three‐ to eight‐fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin‐crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function. Copyright

Formulation and Characterization of Echogenic Lipid–Pluronic Nanobubbles

The advent of microbubble contrast agents has enhanced the capabilities of ultrasound as a medical imaging modality and stimulated innovative strategies for ultrasound-mediated drug and gene delivery. While the utilization of microbubbles as carrier vehicles has shown encouraging results in cancer therapy, their applicability has been limited by a large size which typically confines them to the vasculature. To enhance their multifunctional contrast and delivery capacity, it is critical to reduce bubble size to the nanometer range without reducing echogenicity. In this work, we present a novel strategy for formulation of nanosized, echogenic lipid bubbles by incorporating the surfactant Pluronic, a triblock copolymer of ethylene oxide copropylene oxide coethylene oxide into the formulation. Five Pluronics (L31, L61, L81, L64 and P85) with a range of molecular weights (M(w): 1100 to 4600 Da) were incorporated into the lipid shell either before or after lipid film hydration and before addition of perfluorocarbon gas. Results demonstrate that Pluronic-lipid interactions lead to a significantly reduced bubble size. Among the tested formulations, bubbles made with Pluronic L61 were the smallest with a mean hydrodynamic diameter of 207.9 +/- 74.7 nm compared to the 880.9 +/- 127.6 nm control bubbles. Pluronic L81 also significantly reduced bubble size to 406.8 +/- 21.0 nm. We conclude that Pluronic is effective in lipid bubble size control, and Pluronic M(w), hydrophilic-lipophilic balance (HLB), and Pluronic/lipid ratio are critical determinants of the bubble size. Most importantly, our results have shown that although the bubbles are nanosized, their stability and in vitro and in vivo echogenicity are not compromised. The resulting nanobubbles may be better suited for contrast enhanced tumor imaging and subsequent therapeutic delivery.

Effect of injection site on in situ implant formation and drug release in vivo

In situ forming drug delivery implants offer an attractive alternative to pre-formed implant devices for local drug delivery due to their ability to deliver fragile drugs, simple manufacturing process, and less invasive placement. However, the clinical translation of these systems has been hampered, in part, by poor correlation between in vitro and in vivo drug release profiles. To better understand this effect, the behavior of poly(D,l-lactide-co-glycolide) (PLGA) in situ forming implants was examined in vitro and in vivo after subcutaneous injection as well as injection into necrotic, non-necrotic, and ablated tumor. Implant formation was quantified noninvasively using an ultrasound imaging technique. Drug release of a model drug agent, fluorescein, was correlated with phase inversion in different environments. Results demonstrated that burst drug release in vivo was greater than in vitro for all implant formulations. Drug release from implants in varying in vivo environments was fastest in ablated tumor followed by implants in non-necrotic tumor, in subcutaneous tissue, and finally in necrotic tumor tissue with 50% of the loading drug mass released in 0.7, 0.9, 9.7, and 12.7h respectively. Implants in stiffer ablated and non-necrotic tumor tissue showed much faster drug release than implants in more compliant subcutaneous and necrotic tumor environments. Finally, implant formation examined using ultrasound confirmed that in vivo the process of precipitation (phase inversion) was directly proportional to drug release. These findings suggest that not only is drug release dependent on implant formation but that external environmental effects, such as tissue mechanical properties, may explain the differences seen between in vivo and in vitro drug release from in situ forming implants.

Noninvasive Characterization of In situ Forming Implants Using Diagnostic Ultrasound

In situ forming drug delivery systems provide a means by which a controlled release depot can be physically inserted into a target site without the use of surgery. The release rate of drugs from these systems is often related to the rate of implant formation. Currently, only a limited number of techniques are available to monitor phase inversion, and none of these methods can be used to visualize the process directly and noninvasively. In this study, diagnostic ultrasound was used to visualize and quantify the process of implant formation in a phase inversion based system both in vitro and in vivo. Concurrently, sodium fluorescein was used as a mock drug to evaluate the drug release profiles and correlate drug release and implant formation processes. Implants comprised of three different molecular weight poly(lactic-co-glycolic acid) (PLGA) polymers dissolved in 1-methyl-2-pyrrolidinone (NMP) were studied in vitro and a 29 kDa PLGA solution was evaluated in vivo. The implants were encapsulated in a 1% agarose tissue phantom for five days, or injected into a rat subcutaneously and evaluated for 48 h. Quantitative measurements of the gray-scale value (corresponding to the rate of implant formation), swelling, and precipitation were evaluated using image analysis techniques, showing that polymer molecular weight has a considerable effect on the swelling and formation of the in situ drug delivery depots. A linear correlation was also seen between the in vivo release and depot formation (R(2)=0.93). This study demonstrates, for the first time, that ultrasound can be used to noninvasively and nondestructively monitor and evaluate the phase inversion process of in situ forming drug delivery implants, and that the formation process can be directly related to the initial phase of drug release dependent on this formation.

Characterization of formulation parameters affecting low molecular weight drug release from in situ forming drug delivery systems.

In situ forming implants (ISFI) have shown promise in delivering adjuvant chemotherapy following minimally invasive cancer therapies such as thermal ablation of tumors. Although ISFI systems have been thoroughly investigated for delivery of high molecular weight (Mw) therapeutics, little research has been conducted to optimize their design for delivery of low Mw drugs. This study examined the effect of varying the formulation components on the low Mw drug release profile from a ISFI consisting of poly(D,L-lactide-co-glycolide) (PLGA), fluorescein (model drug), and excipient dissolved in 1-methyl-2-pyrrolidinone (NMP). Effects of varying PLGA Mw, excipient concentration, and drug loading were studied. Additionally, solubility studies were conducted to determine the critical water concentration required for phase inversion. Results demonstrated that PLGA Mw was the most significant factor in modulating low Mw drug release from the ISFI systems. ISFI formulations comprised of a low Mw (16 kDa) PLGA showed a significantly (p < 0.05) lower burst release (after 24 h), 28.2 +/- 0.5%, compared with higher Mw PLGA (60 kDa), 55.1 +/- 3.1%. Critical water concentration studies also demonstrated that formulations with lower Mw PLGA had increased solubility in water and may thus require more time to phase invert and release the drug.

Noninvasive Characterization of the Effect of Varying PLGA Molecular Weight Blends on In Situ Forming Implant Behavior Using Ultrasound Imaging

In situ forming implants (ISFIs) have shown promise in drug delivery applications due to their simple manufacturing and minimally invasive administration. Precise, reproducible control of drug release from ISFIs is essential to their successful clinical application. This study investigated the effect of varying the molar ratio of different molecular weight (Mw) poly(D,L-lactic-co-glycolic acid) (PLGA) polymers within a single implant on the release of a small Mw mock drug (sodium fluorescein) both in vitro and in vivo. Implants were formulated by dissolving three different PLGA Mw (15, 29, and 53kDa), as well as three 1:1 molar ratio combinations of each PLGA Mw in 1-methyl-2-pyrrolidinone (NMP) with the mock drug fluorescein. Since implant morphology and microstructure during ISFI formation and degradation is a crucial determinant of implant performance, and the rate of phase inversion has been shown to have an effect on the implant microstructure, diagnostic ultrasound was used to noninvasively quantify the extent of phase inversion and swelling behavior in both environments. Implant erosion, degradation, as well as the in vitro and in vivo release profiles were also measured using standard techniques. A non-linear mathematical model was used to correlate the drug release behavior with polymer phase inversion, with all formulations yielding an R2 value greater than 0.95. Ultrasound was also used to create a 3D image reconstruction of an implant over a 12 day span. In this study, swelling and phase inversion were shown to be inversely related to the polymer Mw with 53kDa polymer implants increasing at an average rate of 9.4%/day compared with 18.6%/day in the case of the 15 kDa PLGA. Additionally the onset of erosion, complete phase inversion, and degradation facilitated release required 9 d for 53 kDa implants, while these same processes began 3 d after injection into PBS with the 15 kDa implants. It was also observed that PLGA blends generally had intermediate properties when compared to pure polymer formulations. However, release profiles from the blend formulations were governed by a more complex set of processes and were not simply averages of release profiles from the pure polymers preparations. This study demonstrated that implant properties such as phase inversion, swelling and drug release could be tailored to by altering the molar ratio of the polymers used in the depot formulation.

Whole animal imaging.

Translational research plays a vital role in understanding the underlying pathophysiology of human diseases, and hence development of new diagnostic and therapeutic options for their management. After creating an animal disease model, pathophysiologic changes and effects of a therapeutic intervention on them are often evaluated on the animals using immunohistologic or imaging techniques. In contrast to the immunohistologic techniques, the imaging techniques are noninvasive and hence can be used to investigate the whole animal, oftentimes in a single exam which provides opportunities to perform longitudinal studies and dynamic imaging of the same subject, and hence minimizes the experimental variability, requirement for the number of animals, and the time to perform a given experiment. Whole animal imaging can be performed by a number of techniques including x‐ray computed tomography, magnetic resonance imaging, ultrasound imaging, positron emission tomography, single photon emission computed tomography, fluorescence imaging, and bioluminescence imaging, among others. Individual imaging techniques provide different kinds of information regarding the structure, metabolism, and physiology of the animal. Each technique has its own strengths and weaknesses, and none serves every purpose of image acquisition from all regions of an animal. In this review, a broad overview of basic principles, available contrast mechanisms, applications, challenges, and future prospects of many imaging techniques employed for whole animal imaging is provided. Our main goal is to briefly describe the current state of art to researchers and advanced students with a strong background in the field of animal research. Copyright

Effect of cargo properties on in situ forming implant behavior determined by noninvasive ultrasound imaging

Diagnostic ultrasound has been shown to be an effective method for the noninvasive characterization of in situ forming implant behavior both in vivo and in vitro through the evaluation of the echogenic signal that forms as a consequence of the polymer phase transition from liquid to solid. The kinetics of this phase transition have a direct effect on drug release and can be altered through factors that change the mass transfer events of the solvent and aqueous environment, including properties of the entrapped active agent. This study examined the effect of payload properties on implant phase inversion, swelling, drug release, and polymer degradation. Poly(dl-lactide-co-gylcolide) implants were loaded with either: sodium fluorescein, bovine serum albumin (BSA), doxorubicin (Dox), or 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Fluorescein and Dox were released at near equivalent rates throughout the diffusion phase of release but due to differing drug–matrix interactions, Dox-loaded implants released a lower mass of drug during the degradation phase of release. DiI was not readily released, and due to increased depot hydrophobicity, resulted in significantly lower swelling than the other formulations. The initial echogenicity was higher in Dox-loaded implants than those loaded with fluorescein, but after the initial precipitation, phase inversion and drug release occurred at near equivalent rates for both Dox and fluorescein-loaded implants. Nonlinear mathematical fitting was used to correlate drug release and phase inversion, providing a noninvasive method for evaluating implant release (R2 > 0.97 for Dox, BSA, and fluorescein; DiI had a correlation coefficient of 0.56).

Effect of the Subcutaneous Environment on Phase Sensitive In Situ Forming Implant Drug Release, Degradation, and Microstructure

In situ-forming implants are a promising platform used for the release of therapeutic agents. Significant changes in behavior occur when the implants are used in vivo relative to implants formed in vitro. To understand how the injection site effects implant behavior, poly(lactic-co-glycolic acid) implants were examined after injection in the subcutaneous space of a Sprague-Dawley rat model to determine how the environment altered implant erosion, degradation, swelling, microstructure, and mock drug release. Changes in implant microstructure occurred over time for implants formed in vivo, where it was observed that the porosity was lost over the course of 5 days. Implants formed in vivo had a significantly greater burst release (p < 0.05) relative to implants formed in vitro. However, during the diffusion period of release, implants formed in vitro had a significantly higher daily release (2.1%/day, p < 0.05), which correlated to changes in implant microstructure. Additionally, implants formed in vitro had a two-fold increase in the first-order degradation kinetics relative to the implants formed in vivo. These findings suggest that the changes in implant behavior occur as a result of changes in the implant microstructure induced by the external environment.

Differentiation of Benign Periablational Enhancement from Residual Tumor Following Radio-Frequency Ablation Using Contrast-Enhanced Ultrasonography in a Rat Subcutaneous Colon Cancer Model

Benign periablational enhancement (BPE) response to thermal injury is a barrier to early detection of residual tumor in contrast enhanced imaging after radio-frequency (RF) ablation. The objective of this study was to evaluate the role of quantitative of contrast-enhanced ultrasound (CEUS) in early differentiation of BPE from residual tumor in a BD-IX rat subcutaneous colon cancer model. A phantom study was first performed to test the validity of the perfusion parameters in predicting blood flow of two US contrast imaging modes-contrast harmonic imaging (CHI) and microflow imaging (MFI). To create a simple model of BPE, a peripheral portion of the tumor was ablated along with surrounding normal tissue, leaving part of the tumor untreated. First-pass dynamic enhancement (FPDE) and MFI scans of CEUS were performed before ablation and immediately, 1, 4 and 7 days after ablation. Time-intensity-curves in regions of BPE and residual tumor were fitted to the function y = A(1-exp[-β{t-t0}])+C, in which A, β, t0 and C represent blood volume, flow speed, time to start and baseline intensity, respectively. In the phantom study, positive linear correlations were noted between A, β, Aβ and contrast concentration, speed and flow rate, respectively, in both CHI and MFI. On CEUS images of the in vivo study, the unenhanced ablated zone was surrounded by BPE and irregular peripheral enhancement consistent with residual tumor. On days 0, 4 and 7, blood volume (A) in BPE was significantly higher than that in residual tumor in both FPDE imaging and MFI. Significantly greater blood flow (Aβ) was seen in BPE compared with residual tumor tissue in FPDE on day 7 and in MFI on day 4. The results of this study demonstrate that qualitative CEUS can be potentially used for early detection of viable tumor in post-ablation assessment.