Thermoresponsive swelling of photoacoustic single-chain nanoparticles.

NIR-fluorescent LCST-type single-chain nanoparticles (SCNPs) change their photophysical behaviour upon heating, caused by depletion of water from the swollen SCNP interiors. This thermoresponsive effect leads to a fluctuating photoacoustic (PA) signal which can be used as a contrast mechanism for PA imaging.

Luciferase Expressing Preclinical Model Systems Representing the Different Molecular Subtypes of Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease. More insight into the biological diversity of CRC is needed to improve therapeutic outcomes. Established CRC cell lines are frequently used and were shown to be representative models of the main subtypes of CRC at the genomic and transcriptomic level. In the present work, we established stable, luciferase expressing derivatives from 10 well-established CRC cell lines, generated spheroids and subcutaneous xenograft tumors in nude mice, and performed comparative characterization of these model systems. Transcriptomic analyses revealed the close relation of cell lines with their derived spheroids and xenograft tumors. The preclinical model systems clustered with patient tumor samples when compared to normal tissue thereby confirming that cell-line-based tumor models retain specific characteristics of primary tumors. Xenografts showed different differentiation patterns and bioluminescence imaging revealed metastatic spread to the lungs. In addition, the models were classified according to the CMS classification system, with further sub-classification according to the recently identified two intrinsic epithelial tumor cell states of CRC, iCMS2 and iCMS3. The combined data showed that regarding primary tumor characteristics, 3D-spheroid cultures resemble xenografts more closely than 2D-cultured cells do. Furthermore, we set up a bioluminescence-based spheroid cytotoxicity assay in order to be able to perform dose-response relationship studies in analogy to typical monolayer assays. Applying the established assay, we studied the efficacy of oxaliplatin. Seven of the ten used cell lines showed a significant reduction in the response to oxaliplatin in the 3D-spheroid model compared to the 2D-monolayer model. Therapy studies in selected xenograft models confirmed the response or lack of response to oxaliplatin treatment. Analyses of differentially expressed genes in these models identified CAV1 as a possible marker of oxaliplatin resistance. In conclusion, we established a combined 2D/3D, in vitro/in vivo model system representing the heterogeneity of CRC, which can be used in preclinical research applications.

A starch-based implant as a controlled drug release system: Non-invasive in vivo characterization using multispectral fluorescence imaging.

Solid implants are parenteral depot systems that can provide a controlled release of drugs in the desired body area over a few days to months. Finding an alternative for the two most commonly used polymers in the production of parenteral depot systems, namely Poly-(lactic acid) (PLA) and Poly-(lactide-co-glycolide) (PLGA), is of great importance due to their certain drawbacks. Our previous study showed the general suitability of starch-based implants for controlled drug release system. In this study, the system is further characterized and the release kinetics are investigated in vitro and in vivo by fluorescence imaging (FI). ICG and DiR, two fluorescent dyes with different hydrophobicity serving as a model for hydrophilic and hydrophobic drugs, have been used. In addition to 2D FI, 3D reconstructions of the starch implant were also used to assess the release kinetics in 3D mode. The in vitro and in vivo studies showed a fast release of ICG and a sustained release of DiR over 30 days from the starch-based implant. No treatment-related adverse effects were observed in mice. Our results indicate the promising potential of the biodegradable biocompatible starch-based implant for the controlled release of hydrophobic drugs.

Development and Characterization of Novel In-Situ-Forming Oleogels.

PLGA-based in situ forming implants (ISFI) often require a high amount of potentially toxic solvents such as N methyl-Pyrrolidone (NMP). The aim of the present study was to develop lipid in-situ-forming oleogels (ISFOs) as alternative delivery systems. 12-Hydroxystearic acid (12-HSA) was selected as the oleogelling agent and three different oleoformulations were investigated: (a) 12-HSA, peanut oil (PO), NMP; (b) 12-HSA, medium-chain triglycerides (MCT), ethanol; (c) 12-HSA, isopropyl myristate (IPM), ethanol. The effects of the 12-HSA concentration, preparation method, and composition on the mechanical stability were examined using a texture analysis and oscillating rheology. The texture analysis was used to obtain information on the compression strength. The amplitude sweeps were analyzed to provide information on the gel strength and the risk of brittle fractures. The frequency sweeps allowed insights into the long-term stability and risk of syneresis. The syringeability of the ISFOs was tested, along with their acute and long-term cytotoxicity in vitro. The developed ISFOs have the following advantages: (1) the avoidance of highly acidic degradation products; (2) low amounts of organic solvents required; (3) low toxicity; (4) low injection forces, even with small needle sizes. Therefore, ISFOs are promising alternatives to the existing polymer/NMP-based ISFIs.

Development of Poly(sorbitol adipate)-g-poly(ethylene glycol) Mono Methyl Ether-Based Hydrogel Matrices for Model Drug Release.

Hydrogels were prepared by Steglich esterification and by crosslinking pre-synthesized poly(sorbitol adipate)-graft-poly(ethylene glycol) mono methyl ether (PSA-g-mPEG) using different-chain-length-based disuccinyl PEG. PSA and PSA-g-mPEG were investigated for polymer degradation as a function of time at different temperatures. PSA-g-mPEG hydrogels were then evaluated for their most crucial properties of swelling that rendered them suitable for many pharmaceutical and biomedical applications. Hydrogels were also examined for their Sol-Gel content in order to investigate the degree of cross-linking. Physical structural parameters of the hydrogels were theoretically estimated using the modified Flory-Rehner theory to obtain approximate values of polymer volume fraction, the molecular weight between two crosslinks, and the mesh size of the hydrogels. X-ray diffraction was conducted to detect the presence or absence of crystalline regions in the hydrogels. PSA-g-mPEG hydrogels were then extensively examined for higher and lower molecular weight solute release through analysis by fluorescence spectroscopy. Finally, the cytotoxicity of the hydrogels was also investigated using a resazurin reduction assay. Experimental results show that PSA-g-mPEG provides an option as a biocompatible polymer to be used for pharmaceutical applications.

Particle Engineering of Innovative Nanoemulsion Designs to Modify the Accumulation in Female Sex Organs by Particle Size and Surface Charge.

Particle engineering of nanosized drug delivery systems (DDS) can be used as a strategic tool to influence their pharmacokinetics after intravenous (i.v.) application by the targeted adaptation of their particle properties according to the needs at their site of action. This study aimed to investigate particle properties depending on patterns in the biodistribution profile to modify the accumulation in the female sex organs using tailor-made nanoemulsion designs and thereby to either increase therapeutic efficiency for ovarian dysfunctions and diseases or to decrease the side effects caused by unintended accumulation. Through the incorporation of the anionic phospholipid phosphatidylglycerol (PG) into the stabilizing macrogol 15 hydroxystearate (MHS) layer of the nanoemulsions droplets, it was possible to produce tailor-made nanoparticles with tunable particle size between 25 to 150 nm in diameter as well as tunable surface charges between -2 to nearly -30 mV zeta potential using a phase inversion-based process. Three chosen negatively surface-charged nanoemulsions of 50, 100, and 150 nm in diameter showed very low cellular toxicities on 3T3 and NHDF fibroblasts and merely interacted with the blood cells, but instead stayed inert in the plasma. In vivo and ex vivo fluorescence imaging of adult female mice i.v. injected with the negatively surface-charged nanoemulsions revealed a high accumulation depending on their particle size in the reticuloendothelial system (RES), being found in the liver and spleen with a mean portion of the average radiant efficiency (PARE) between 42-52%, or 8-10%, respectively. With increasing particle size, an accumulation in the heart was detected with a mean PARE up to 8%. These three negatively surface-charged nanoemulsions overcame the particle size-dependent accumulation in the female sex organs and accumulated equally with a small mean PARE of 5%, suitable to reduce the side effects caused by unintended accumulation while maintaining different biodistribution profiles. In contrast, previously investigated neutral surface-charged nanoemulsions accumulated with a mean PARE up to 10%, strongly dependent on their particle sizes, which is useful to improve the therapeutic efficacy for ovarian dysfunctions and diseases.

Imidazole Analogs of Vascular-Disrupting Combretastatin A-4 with Pleiotropic Efficacy against Resistant Colorectal Cancer Models.

Specific targeting of the tumoral vasculature by vascular-disrupting agents (VDA), of which combretastatin A-4 (CA-4) is a main representative, has been considered a new therapeutic strategy against multidrug-resistant tumors. In addition, CA-4 and analogs are tubulin-targeting agents and can exert direct antitumor effects by different mechanisms. Herein, we analyzed a series of synthetic CA-4 analogs featuring N-methylimidazole-bridged Z-alkenes with different halo- or amino-substituted aryl rings in vitro and in vivo, focusing on models of colorectal cancer. Combined in vitro/in vivo structure-activity relationship studies using cell lines and xenograft tumors susceptible to VDA-induced vascular damage demonstrated a clear association of cytotoxic and vascular-disrupting activity with the ability to inhibit tubulin polymerization, which was determined by specific substitution constellations. The most active compounds were tested in an extended panel of colorectal cancer (CRC) cell lines and showed activity in CA-4-resistant and chemotherapy-resistant cell lines. The bromo derivative brimamin was then compared with the known fosbretabulin (CA-4P) by activity tests on DLD-1- (multidrug-resistant) and HT29- (CA-4-resistant) derived xenograft tumors. Treatment did not induce pronounced vascular-disrupting effects in these tumors. Histological analyses revealed distinct tumor substructures and vessel compositions of DLD-1/HT29 tumors, which clearly differed from the tumor models susceptible to VDA treatment. Even so, brimamin effectively retarded the growth of DLD-1 tumors, overcoming their resistance to standard treatment, and it inhibited the outgrowth of disseminated HT29 tumor cells in an experimental metastasis model. In conclusion, combretastatin analogous N-methylimidazoles proved capable of inducing vascular-disrupting effects, comparable to those of CA-4P. In addition, they showed antitumor activities in models of drug-resistant colorectal cancer, independent of vascular-disrupting effects.

Selection and Incorporation of siRNA Carrying Non-Viral Vector for Sustained Delivery from Gellan Gum Hydrogels.

The local controlled release of siRNA is an attractive and rational strategy to enhance and extend the effectiveness of gene therapy. Since naked and unmodified siRNA has a limited cell uptake and knockdown efficiency, the complexation of siRNA with non-viral carriers is often necessary for the delivery of bioactive RNA. We evaluated the performance of three different non-viral siRNA carriers, including DOTAP lipoplexes (DL), chitosan polyplexes (CP), and solid lipid complexes (SLC). The physicochemical properties of the siRNA-nanocarriers were characterized by dynamic light scattering and gel electrophoresis. After in vitro characterization, the carrier with the most appropriate properties was found to be the DL suspension, which was subsequently loaded into a gellan gum hydrogel matrix and examined for its drug load, stability, and homogeneity. The hydrogels microstructure was investigated by rheology to assess the impact of the rheological properties on the release of the siRNA nanocarriers. A controlled release of complexed siRNA over 60 days in vitro was observed. By comparing the results from fluorescence imaging with data received from HPLC measurements, fluorescence imaging was found to be an appropriate tool to measure the release of siRNA complexes. Finally, the bioactivity of the siRNA released from hydrogel was tested and compared to free DL for its ability to knockdown the GFP expression in a DLD1 colon cancer cell model. The results indicate controlled release properties and activity of the released siRNA. In conclusion, the developed formulation is a promising system to provide local controlled release of siRNA over several weeks.

Ovarian Accumulation of Nanoemulsions: Impact of Mice Age and Particle Size.

Nanotechnology in the field of drug delivery comes with great benefits due to the unique physicochemical properties of newly developed nanocarriers. However, they may come as well with severe toxicological side effects because of unwanted accumulation in organs outside of their targeted site of actions. Several studies showed an unintended accumulation of various nanocarriers in female sex organs, especially in the ovaries. Some led to inflammation, fibrosis, or decreasing follicle numbers. However, none of these studies investigated ovarian accumulation in context to both reproductive aging and particle size. Besides the influences of particle size, the biodistribution profile may be altered as well by reproductive aging because of reduced capacities of the reticuloendothelial system (RES), changes in sex steroid hormone levels as well as altering ovarian stromal blood flow. This systematic investigation of the biodistribution of intravenously (i.v) injected nanoemulsions revealed significant dependencies on the two parameters particle size and age starting from juvenile prepubescent to senescent mice. Using fluorescent in vivo and ex vivo imaging, prepubescent mice showed nearly no accumulation of nanoemulsion in their uteri and ovaries, but high accumulations in the organs of the RES liver and spleen independently of the particle size. In fertile adult mice, the accumulation increased significantly in the ovaries with an increased particle size of the nanoemulsions by nearly doubling the portion of the average radiant efficiency (PARE) to ~10% of the total measured signal of all excised organs. With reproductive aging and hence loss of fertility in senescent mice, the accumulation decreased again to moderate levels, again independently of the particle size. In conclusion, the ovarian accumulation of these nanocarriers depended on both the age plus the particle size during maturity.

Towards the Development of Long Circulating Phosphatidylserine (PS)- and Phosphatidylglycerol (PG)-Enriched Anti-Inflammatory Liposomes: Is PEGylation Effective?

The anionic phospholipids (PLs) phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with anti-inflammatory and immunomodulatory activity. A potential clinical use requires well-defined systems and for several applications, a long circulation time is desirable. Therefore, we aimed the development of long circulating liposomes with intrinsic anti-inflammatory activity. Hence, PS- and PG-enriched liposomes were produced, whilst phosphatidylcholine (PC) liposomes served as control. Liposomes were either formulated as conventional or PEGylated formulations. They had diameters below 150 nm, narrow size distributions and composition-dependent surface charges. Pharmacokinetics were assessed non-invasively via in vivo fluorescence imaging (FI) and ex vivo in excised organs over 2 days. PC liposomes, conventionally formulated, were rapidly cleared from the circulation, while PEGylation resulted in prolongation of liposome circulation robustly distributing among most organs. In contrast, PS and PG liposomes, both as conventional or PEGylated formulations, were rapidly cleared. Non-PEGylated PS and PG liposomes did accumulate almost exclusively in the liver. In contrast, PEGylated PS and PG liposomes were observed mainly in liver and spleen. In summary, PEGylation of PS and PG liposomes was not effective to prolong the circulation time but caused a higher uptake in the spleen.

Intratumoral Distribution and pH-Dependent Drug Release of High Molecular Weight HPMA Copolymer Drug Conjugates Strongly Depend on Specific Tumor Substructure and Microenvironment.

Stimulus-sensitive polymer drug conjugates based on high molecular weight N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers carrying doxorubicin via a pH-dependent cleavable bond (pHPMA-Dox) were previously shown to be able to overcome multi-drug resistance. Nevertheless, a tumor type dependent differential response was observed. Although an improved and more selective tumor accumulation of pHPMA-Dox is generally achieved due to the enhanced permeability and retention (EPR) effect, little is known about the fate of these conjugates upon entering the tumor tissue, which could explain the different responses. In this study, we compared in vitro and in vivo accumulation and Dox-activation of pHPMA-Dox in three cancer cell line models (1411HP, A2780cis, HT29) and derived xenograft tumors using a near-infrared fluorescence-labeled pHPMA-Dox conjugate. Firstly, cytotoxicity assays using different pH conditions proved a stepwise, pH-dependent increase in cytotoxic activity and revealed comparable sensitivity among the cell lines. Using multispectral fluorescence microscopy, we were able to track the distribution of drug and polymeric carrier simultaneously on cellular and histological levels. Microscopic analyses of cell monolayers confirmed the assumed mechanism of cell internalization of the whole conjugate followed by intracellular cleavage and nuclear accumulation of Dox in all three cell lines. In contrast, intratumoral distribution and drug release in xenograft tumors were completely different and were associated with different tissue substructures and microenvironments analyzed by Azan- and Hypoxisense®-staining. In 1411HP tumors, large vessels and less hypoxic/acidic microenvironments were associated with a pattern resulting from consistent tissue distribution and cellular uptake as whole conjugate followed by intracellular drug release. In A2780cis tumors, an inconsistent pattern of distribution partly resulting from premature drug release was associated with a more hypoxic/acidic microenvironment, compacted tumor tissue with compressed vessels and specific pre-damaged tissue structures. A completely different distribution pattern was observed in HT29 tumors, resulting from high accumulation of polymer in abundant fibrotic structures, with small embedded vessels featuring this tumor type together with pronounced premature drug release due to the strongly hypoxic/acidic microenvironment. In conclusion, the pattern of intratumoral distribution and drug release strongly depends on the tumor substructure and microenvironment and may result in different degrees of therapeutic efficacy. This reflects the pronounced heterogeneity observed in the clinical application of nanomedicines and can be exploited for the future design of such conjugates.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) enriched mixed micelles (MM): A new nano-drug delivery system with anti-inflammatory potential?

Phosphatidylserine (PS) and phosphatidylglycerol (PG) are naturally occurring phospholipids (PL) with intrinsic anti-inflammatory properties. The therapeutic potential of PS and PG has not been extensively explored and the main focus had been directed towards PS- and PG-liposomes. In order to increase the formulation options, we explored whether mixed micelles (MM) could be an alternative to liposomes. Potential advantages of MM are their thermodynamic stability, small size and ease of manufacture. DOPS- and DOPG-enriched MM were obtained via a co-precipitation technique and physicochemical characterization was performed. The MM, approximately 10 nm in diameter, showed no toxicity on fibroblast cell lines in vitro and virtually no hemolytic activity. The MM suppressed the TNFα-production of mIFNγ/LPS-stimulated mouse peritoneal macrophages (MPM) in vitro similar to DOPS- and DOPG-liposomes. Therefore, DOPS- and DOPG-loaded MM are promising new options for the treatment of inflammatory diseases.

Phase inversion-based nanoemulsions of medium chain triglyceride as potential drug delivery system for parenteral applications.

Lipid nanoemulsions are attractive drug delivery systems for lipophilic drugs. To produce nanoemulsions with droplets of very small diameter (<100 nm), we investigated thermotropic phase transitions as an alternative to the standard procedure of high-pressure homogenization. Employing shock dilution with ice-cold water during the phase inversion gives the opportunity to produce nanoemulsions without any use of potentially toxic organic solvents. The systematic investigation of the relation of the three involved components surfactant, aqueous phase and lipid phase showed that depending on the ratio of surfactant to lipid the emulsions contained particles of diameters between 16 and 175 nm with narrow polydispersity index distributions and uncharged surfaces. Nanoemulsions with particles of 50 and 100 nm in diameter showed very little toxicity to fibroblast cells in vitro. An unusual, exponential-like nonlinear increase in osmolality was observed with increasing concentration of the nonionic surfactant Kolliphor HS 15. The experimental results indicate, that nanoemulsions with particles of small and tunable size can be easily formed without homogenization by thermal cycling.

In situ Gelling Amphotericin B Nanofibers: A New Option for the Treatment of Keratomycosis.

The purpose of our research was the development of Amphotericin B-loaded in situ gelling nanofibers for the treatment of keratomycosis. Different formulation strategies were applied to increase the drug load of the sparingly water-soluble Amphotericin B in electrospun Gellan Gum/Pullulan fibers. These include bile salt addition, encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and formation of a polymeric Amphotericin B polyelectrolyte complex. The Amphotericin B polyelectrolyte complex (AmpB-Eu L) performed best and was very effective against the fungal strain Issatchenkia orientalis in vitro. The complex was characterized in detail by attenuated total reflection infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry. A heat induced stress test was carried out to ensure the stability of the polyelectrolyte complex. To gain information about the cellular tolerance of the developed polyelectrolyte complex a new, innovative multilayered-stratified human cornea cell model was used for determination of the cellular toxicity in vitro. For a safe therapy, the applied ophthalmic drug delivery system has to be sterile. Sterilization by electron irradiation caused not degradation of pure Amphotericin B and also for the bile salt complex. Furthermore, the developed Amphotericin B polyelectrolyte complex was not degraded by the irradiation process. In conclusion, a new polyelectrolyte Amphotericin B complex has been found which retains the antifungal activity of the drug with sufficient stability against irradiation-sterilization induced drug degradation. Furthermore, in comparison with the conventional used eye drop formulation, the new AmpB-complex loaded nanofibers were less toxic to cornea cells in vitro. Electrospinning of the Amphotericin B polyelectrolyte complex with Gellan Gum/ Pullulan leads to the formation of nanofibers with in situ gelling properties, which is a new and promising option for the treatment of keratomycosis.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) nanodispersions as potential anti-inflammatory therapeutics: Comparison of in vitro activity and impact of pegylation.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with putative anti-inflammatory potential. However, studies comparing PS and PG are rare and were mainly conducted with phospholipid-dispersions of large size and broad distributions. Thus, we prepared small-sized PS- and PG-loaded liposomes exhibiting narrow distribution, and additionally studied the impact of liposome-pegylation on the reduction of the TNFα-production caused by the PS- and PG-liposomes. These PS- and PG-containing nanodispersions had a small size around 100nm and a narrow distribution (PDI<0.1). The liposome-dispersions showed no toxicity in NHDF- and 3T3-cells and virtually no hemolytic activity. They decreased the TNFα-production of LPS-(lipopolysaccharide)-stimulated mouse peritoneal macrophages in vitro. PG-liposomes always decreased the TNFα-levels more potently than PS-liposomes. Pegylation of PS- and PG-liposomes caused different Zeta potentials, but did not change biological activity. The results of the current study indicate a high potential of the tested formulations for phospholipid-based anti-inflammatory therapies.

Efficacy of a Bispecific Antibody Co-Targeting VEGFA and Ang-2 in Combination with Chemotherapy in a Chemoresistant Colorectal Carcinoma Xenograft Model.

Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. However, anti-angiogenic tumor therapy targeting a single factor may be limited by complementary mechanisms. Angiopoietin-2 (Ang-2, ANGPT2) is another important factor that cooperates with VEGF to drive tumor angiogenesis. It was shown that high Ang-2 levels are associated with a poor clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Therefore, combined inhibition of VEGF and Ang-2 was supposed to improve anti-angiogenic therapy. Here, we evaluated the efficacy of a bispecific antibody (CrossMab) co-targeting VEGF and Ang-2 in combination with chemotherapy in a chemoresistant colorectal carcinoma model. Antitumor activity was evaluated in athymic nude mice bearing subcutaneous DLD1 xenograft tumors and treated with anti-VEGF (B20), anti-Ang-2 (LC06) and anti-VEGF/Ang-2 (CrossMab) antibodies. Chemotherapy consisted of 5-FU and irinotecan. Resected tumors were analyzed immunohistochemically. First, an impact of targeting each single factor but also a clear advantage of co-targeting both factors could be demonstrated. Accordingly, tumor tissue showed strong staining for VEGF and Ang-2. Chemotherapy alone was less effective. Efficient tumor growth inhibition could be achieved by treatment with anti-VEGF/chemotherapy, single CrossMab and CrossMab/chemotherapy, which resulted in 3 out of 10, 6 out of 10 and 10 out of 10 complete responses, respectively, during seven weeks. Complete retarded tumors were characterized by massive intratumoral necrosis surrounded by layers of vital tumor cells and connective tissue with CD31-positive vessels at the periphery. In some cases, a distinct feature known as vessel co-option could be observed. In conclusion, the data from this model clearly support the strategy of co-targeting VEGF and Ang-2 and further demonstrate the beneficial impact of co-treatment with chemotherapy. The clear superiority of the CrossMab-containing regimen compared to clinical standard anti-VEGF/chemotherapy warrants further analyses in other models.

Dual dye in-vivo imaging of differentially charged PLGA carriers reveals antigen-depot effect, leading to improved immune responses in preclinical models.

The present in-vivo study investigated the behavior and performance of differently charged poly(lactic­co­glycolic) acid microparticles (PLGA MP) as vaccination platform. For this purpose, particles loaded with ovalbumin (OVA) as model antigen were subcutaneously (s.c.) injected in SKH1 mice. The utilized SKH1 hairless mice exhibit a fully operative immune system and allow parallel imaging investigations due to the lack of hair. Usage of this species enabled the combination of two investigations within a single study protocol, namely noninvasive in-vivo imaging and immune responses directed towards the antigen. All treatments were well tolerated, no safety drop-outs occurred. The fate of the model antigen OVA as well as the PLGA particles was monitored using a dual dye approach (CF660C & DiR) by multispectral fluorescence imaging (msFI). A depot effect for the OVA antigen adsorbed to the MP surface could be observed for the positively charged MPs. The immune response against OVA was then analyzed. OVA alone did not induce an immune response, whereas the positively charged as well as the neutral MP induced a strong and consistent humoral immune response with a clear favor of IgG1 over IgG2a subclass antibodies. In contrast, negatively charged MP were not able to induce measurable antibody responses. Cellular immune response was weak and inconsistent for all treated groups, which verifies previous in-vitro results conducted with the herein described microparticulate antigen platform. In conclusion, the characterization of the in-vivo performance yielded valuable information about antigen and carrier fate after application. The presented adjuvant platform is capable of inducing strong TH2 dominated immune responses characterized by enhanced IgG1 subclass titers which are critical for vaccines aimed at promoting induction of neutralizing antibodies.

Intended and Unintended Targeting of Polymeric Nanocarriers: The Case of Modified Poly(glycerol adipate) Nanoparticles.

Biodegradable nanoparticles based on stearic acid-modified poly(glycerol adipate) (PGAS) are promising carriers for drug delivery. In order to investigate the impact of the particle interface characteristics on the biological fate, PGAS nanoparticles are covalently and noncovalently coated with N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. HPMA copolymer-modified PGAS nanoparticles have similar particle sizes, but less negative zeta-potentials. Nanoparticles are double labeled with the fluorescent dyes DiR (noncovalently) and DYOMICS-676 (covalently bound to HPMA copolymer), and their biodistribution is investigated noninvasively by multispectral optical imaging. Both covalent and noncovalent coatings cause changes in the pharmacokinetics and biodistribution in healthy and tumor-bearing mice. In addition to the intended tumor accumulation, high signals of both fluorescent dyes are also observed in other organs, including liver, ovaries, adrenal glands, and bone. The unintended accumulation of nanocarriers needs further detailed and systematic investigations, especially with respect to the observed ovarian and adrenal gland accumulation.

Biodegradable Micellar HPMA-Based Polymer-Drug Conjugates with Betulinic Acid for Passive Tumor Targeting.

Here, we present the synthesis, physicochemical, and preliminary biological characterization of micellar polymer-betulinic acid (BA) conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carriers, enabling the controlled release of cytotoxic BA derivatives in solid tumors or tumor cells. Various HPMA copolymer conjugates differing in the structure of the spacer between the drug and the carrier were synthesized, all designed for pH-triggered drug release in tumor tissue or tumor cells. The high molecular weight of the micellar conjugates should improve the uptake of the drug in solid tumors due to the Enhanced permeability and retention (EPR) effect. Nevertheless, only the conjugate containing BA with methylated carboxyl groups enabled pH-dependent controlled release in vitro. Moreover, drug release led to the disassembly of the micellar structure, which facilitated elimination of the water-soluble HPMA copolymer carrier from the body by renal filtration. The methylated BA derivative and its polymer conjugate exhibited high cytostatic activity against DLD-1, HT-29, and HeLa carcinoma cell lines and enhanced tumor accumulation in HT-29 xenograft in mice.

Improved Tumor-Specific Drug Accumulation by Polymer Therapeutics with pH-Sensitive Drug Release Overcomes Chemotherapy Resistance.

The success of chemotherapy is limited by poor selectivity of active drugs combined with occurrence of tumor resistance. New star-like structured N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based drug delivery systems containing doxorubicin attached via a pH-sensitive hydrazone bond were designed and investigated for their ability to overcome chemotherapy resistance. These conjugates combine two strategies to achieve a high drug concentration selectively at the tumor site: (I) high accumulation by passive tumor targeting based on enhanced permeability and retention effect and (II) pH-sensitive site-specific drug release due to an acidic tumor microenvironment. Mice bearing doxorubicin-resistant xenograft tumors were treated with doxorubicin, PBS, poly HPMA (pHPMA) precursor or pHPMA-doxorubicin conjugate at different equivalent doses of 5 mg/kg bodyweight doxorubicin up to a 7-fold total dose using different treatment schedules. Intratumoral drug accumulation was analyzed by fluorescence imaging utilizing intrinsic fluorescence of doxorubicin. Free doxorubicin induced significant toxicity but hardly any tumor-inhibiting effects. Administering at least a 3-fold dose of pHPMA-doxorubicin conjugate was necessary to induce a transient response, whereas doses of about 5- to 6-fold induced strong regressions. Tumors completely disappeared in some cases. The onset of response was differential delayed depending on the tumor model, which could be ascribed to distinct characteristics of the microenvironment. Further fluorescence imaging-based analyses regarding underlying mechanisms of the delayed response revealed a related switch to a more supporting intratumoral microenvironment for effective drug release. In conclusion, the current study demonstrates that the concept of tumor site-restricted high-dose chemotherapy is able to overcome therapy resistance. Mol Cancer Ther; 15(5); 998-1007. ©2016 AACR.