Optimizing Nanosuspension Drug Release and Wound Healing Using a Design of Experiments Approach: Improving the Drug Delivery Potential of NDH-4338 for Treating Chemical Burns.

NDH-4338 is a highly lipophilic prodrug comprising indomethacin and an acetylcholinesterase inhibitor. A design of experiments approach was used to synthesize, characterize, and evaluate the wound healing efficacy of optimized NDH-4338 nanosuspensions against nitrogen mustard-induced skin injury. Nanosuspensions were prepared by sonoprecipitation in the presence of a Vitamin E TPGS aqueous stabilizer solution. Critical processing parameters and material attributes were optimized to reduce particle size and determine the effect on dissolution rate and burn healing efficacy. The antisolvent/solvent ratio (A/S), dose concentration (DC), and drug/stabilizer ratio (D/S) were the critical sonoprecipitation factors that control particle size. These factors were subjected to a Box-Behnken design and response surface analysis, and model quality was assessed. Maximize desirability and simulation experiment optimization approaches were used to determine nanosuspension parameters with the smallest size and the lowest defect rate within the 10-50 nm specification limits. Optimized and unoptimized nanosuspensions were prepared and characterized. An established depilatory double-disc mouse model was used to evaluate the healing of nitrogen mustard-induced dermal injuries. Optimized nanosuspensions (A/S = 6.2, DC = 2% w/v, D/S = 2.8) achieved a particle size of 31.46 nm with a narrow size range (PDI = 0.110) and a reduced defect rate (42.2 to 6.1%). The optimized nanosuspensions were stable and re-dispersible, and they showed a ~45% increase in cumulative drug release and significant edema reduction in mice. Optimized NDH-4338 nanosuspensions were smaller with more uniform sizes that led to improved physical stability, faster dissolution, and enhanced burn healing efficacy compared to unoptimized nanosuspensions.

Depilatory double-disc mouse model for evaluation of vesicant dermal injury pharmacotherapy countermeasures.

BACKGROUND: Sulfur mustard (SM) is a chemical warfare vesicant that severely injures exposed eyes, lungs, and skin. Mechlorethamine hydrochloride (NM) is widely used as an SM surrogate. This study aimed to develop a depilatory double-disc (DDD) NM skin burn model for investigating vesicant pharmacotherapy countermeasures. METHODS: Hair removal method (clipping only versus clipping followed by a depilatory), the effect of acetone in the vesicant administration vehicle, NM dose (0.5-20 µmol), vehicle volume (5-20 µl), and time course (0.5-21 days) were investigated using male and female CD-1 mice. Edema, an indicator of burn response, was assessed by biopsy skin weight. The ideal NM dose to induce partial-thickness burns was assessed by edema and histopathologic evaluation. The optimized DDD model was validated using an established reagent, NDH-4338, a cyclooxygenase, inducible nitric oxide synthase, and acetylcholinesterase inhibitor prodrug. RESULTS: Clipping/depilatory resulted in a 5-fold higher skin edematous response and was highly reproducible (18-fold lower %CV) compared to clipping alone. Acetone did not affect edema formation. Peak edema occurred 24-48 h after NM administration using optimized dosing methods and volume. Ideal partial-thickness burns were achieved with 5 µmol of NM and responded to treatment with NDH-4338. No differences in burn edematous responses were observed between males and females. CONCLUSION: A highly reproducible and sensitive partial-thickness skin burn model was developed for assessing vesicant pharmacotherapy countermeasures. This model provides clinically relevant wound severity and eliminates the need for organic solvents that induce changes to the skin barrier function.

Breast intraductal nanoformulations for treating ductal carcinoma in situ II: Dose de-escalation using a slow releasing/slow bioconverting prodrug strategy.

Ductal carcinoma in situ (DCIS) represents approximately 20-25% of newly diagnosed breast cancers. DCIS is treated by surgery and possibly radiotherapy. Chemotherapy is only used as adjuvant or neoadjuvant therapy but not as primary therapy. The present study investigated the intraductal administration of Ciclopirox (CPX) formulated in nanosuspensions (NSs) or nanoparticles (NPs) to treat DCIS locally in a Fischer 344 rat model orthotopically implanted with 13762 Mat B III cells. Slow converting esterase responsive CPX prodrugs (CPDs) were successfully synthesized at high purity (> 95%) by directly acetylating the hydroxyl group or by appending a self-immolative linker between CPX and a phenolic ester. Direct esterification CPDs were not sufficiently stable so self-immolative CPDs were formulated in NSs and NPs. Prodrug release was evaluated from poly(lactic-co-glycolic acid) NPs, and CPD4 demonstrated the slowest release rate with the rank order of CPD2 (R = methyl) > CPD3 (R = t-butyl) > CPD4 (R = phenyl). Intraductally administered CPX NS, CPD4 NS, and an innovative mixture of CDP4 NS and NPs (at 1 mg CPX equivalent/duct) demonstrated significant (p < 0.05) in vivo anti-tumor efficacy compared with immediate release (IR) CPX NS and non-treated controls. CPX mammary persistence at 6 h and 48 h after CPD4 NS or NP administration was also greater than after the immediate release CPX NS. A strong correlation between CPX mammary persistence and efficacy is demonstrated. In conclusion, nanoformulations utilizing a slow releasing/slow bioconverting CPX prodrug delivery strategy resulted in significant dose de-escalation (~ five fold) while maintaining anti-tumor efficacy.

Unusual Cu-Co/GO Composite with Special High Organic Content Synthesized by an in Situ Self-Assembly Approach: Pyrolysis and Catalytic Decomposition on Energetic Materials.

An interesting Cu-Co/GO composite with special high organic content was accidentally fabricated for the first time via a one-pot solvothermal method in the mixed solvent of isopropanol and glycerol. The Cu-Co/GO composite was calcined separately in three different atmospheres (air, nitrogen, and argon) and further investigated by a series of characterization techniques. The results indicate that the spinel phase nano-CuCo2O4 composite, nanometal oxides (CuO and CoO), and nanometal mixture of Cu and Co were unexpectedly formed after calcination in air, N2, and Ar atmospheres, respectively, and the possible reaction mechanism was discussed. The specific mass losses of the Cu-Co/GO composite calcined in air, N2, and Ar atmospheres were 28.14 %, 21.68 %, and 23.76 %, respectively. The catalytic decomposition performances of the as-prepared samples for cyclotrimethylenetrinitramine (RDX) and the mixture of nitrocellulose (NC) and RDX (NC + RDX) were investigated and compared via DSC method, and the results demonstrate that Cu-Co/GO composites obviously decrease the thermal decomposition temperature of RDX from 242.3 to 236.5 (before calcination), 238.6 (air), 235.8 (N2), and 228.6 °C (Ar), respectively. Cu-Co/GO(Ar) composite exhibits the best catalytic decomposition performance among all samples, which makes the decomposition temperature of RDX and NC + RDX decrease by 13.7 and 4.9 °C and the apparent activation energy of decomposition for RDX decrease by 110.1 kJ/mol. The enhanced catalytic performance of Cu-Co/GO(Ar) composite could be attributed to the smaller particle size, better crystallinity, and specific well-dispersed metal atoms, whereas the Cu-Co/GO(air) composite after air calcination presents a bad catalytic performance due to the removal of GO.

Breast intraductal nanoformulations for treating ductal carcinoma in situ I: Exploring metal-ion complexation to slow ciclopirox release, enhance mammary persistence and efficacy.

INTRODUCTION: Ductal Carcinoma In Situ (DCIS) represents a significant fraction (~20-25%) of all newly diagnosed breast cancer cases and, if left untreated, a significant fraction of patients will progress to invasive disease. Surgery is the only treatment option. Ciclopirox (CPX), an FDA-approved antifungal drug, has exhibited promising antitumor activity by down-regulating the expression of vital antiapoptotic cellular proteins and inhibiting the genetic expression of several oncogenic pathways. In this study, the feasibility of using nanoscale delivery systems to control release and prolong mammary tissue persistence of a lipophilic metal complex of CPX and Zinc (CPXZn) after intraductal administration was investigated. METHODS: CPX and CPX-Zn nanosuspensions (NSs) were prepared using an evaporative nanoprecipitation-ultra-sonication method. Flash nanoprecipitation was used to prepare PLGA nanoparticles (NPs) loaded with CPXZn. Our established orthotopic DCIS rat model was used to evaluate efficacy. Briefly, two days after 13762 Mat B III cell intraductal inoculation, rats were divided into treatment groups and a single intraductal injection of CPX NS, CPX-Zn NS or CPX-Zn NPs was administered. In the first study arm, the efficacy of CPX NS (1, 3, 5 mg/duct) was evaluated. In the second arm, the in vivo efficacy of CPX NS, CPX-Zn NS and CPX-Zn loaded NPs was evaluated and compared at equivalent CPX doses. The mammary persistence of CPX from CPX NS, CPX-Zn NS, and CPX-Zn PLGA NPs was also assessed. RESULTS: CPX-Zn complex was successfully synthesized and characterized by several spectral analyses. CPX release was slowed from the CPX-Zn NS and further slowed by incorporating CPX-Zn into PLGA NPs as compared to the CPX NS with release half times following the order: CPX NS < CPX-Zn NS << CPX-Zn NP. Intraductal CPX NS administration was dose and time dependent in suppressing tumor initiation suggesting prolonged mammary exposure may improve efficacy. In the second arm, mammary tissue persistence of CPX followed the rank order CPX NS < CPX-Zn NS << CPX-Zn NP at 6 h and 48 h post-administration. Prolonged mammary CPX exposure was highly correlated to improved efficacy. Prolonged CPX tissue persistence, attributed to slower release from the zinc complex and the PLGA NPs, resulted in a 5-fold dose reduction compared to the CPX NS. CONCLUSIONS: The current results demonstrate that slowing drug release in the mammary duct after intraductal administration overcomes the rapid ductal clearance of CPX, prolongs mammary tissue persistence, improves efficacy against DCIS lesions in vivo, and requires 5-fold less CPX to achieve equivalent efficacy. The studies also provide a strategic path forward for developing a locally administered drug delivery system for treating DCIS, for which no primary chemotherapy option is available.

A Novel Bivalent Mannosylated Targeting Ligand Displayed on Nanoparticles Selectively Targets Anti-Inflammatory M2 Macrophages.

Persistent activation of macrophages (MP)s into a proinflammatory M1 or anti-inflammatory M2 phenotype plays a role in several pathological conditions, including autoimmune diseases, fibrosis, infections, atherosclerosis and tumor development. The mannose receptor (MR, CD206), expressed at low levels on resting MPs and absent on M1 MPs, is highly expressed on M2 MPs, making it a potential target and drug delivery portal. Recently, we developed a novel, highly selective MR targeting ligand (MRTL), consisting of two mannose molecules separated by a monodisperse 12 unit poly(ethylene glycol) linker, to enhance the cellular uptake of polymeric nanocarriers. The feasibility of using the MRTL ligand for selectively targeting M2 MPs for intracellular delivery of nanoparticles (NPs) was investigated. Rat peritoneal MPs were differentiated into an M1 or M2 phenotype using IFN-γ and IL-4/IL-13, respectively. Expression of the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 markers arginase (Arg)-1 and MR (at both the mRNA and protein levels) confirmed MP phenotypic activation. Resting, M1 and M2 MPs were treated with fluorescein isothiocyanate (FITC)-labeled MRTL or NPs displaying FITC-labeled MRTL at two surface densities (1 and 10%) and examined by confocal microscopy. Intracellular fluorescence was also quantified. Uptake of the MRTL was 2.4- and 11.8-fold higher in M2 MPs when compared to resting or M1 MPs, respectively, consistent with marker expression levels. Mannan, a competitive inhibitor of the MR, abrogated MRTL uptake. MRTL also co-localized with a fluid-phase endocytosis marker, further suggesting that uptake was mediated by MR-mediated endocytosis. Intracellular NP fluorescence was confirmed by flow cytometry and by confocal microscopy. MRTL-NPs accumulated intracellularly with no significant cell surface binding, suggesting efficient translocation. NPs displaying a low surface density (1%) of the MRTL exhibited significantly higher (2.3-fold) uptake into M2 MPs, relative to resting and M1 MPs. The 10% MRTL-NPs displayed greater uptake by M2 MPs when compared to resting and M1 MPs, but less uptake than 1% MRTL-NPs into M2 MPs. Control FITC-labeled plain NPs did not exhibit selective MP uptake. These studies demonstrate that M2 MPs are selectively targeted by NPs displaying a novel bivalent ligand that utilizes the MR as a target/portal for cell entry. This study also establishes the feasibility of the approach allowing for further investigation in vivo.

Correction: CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

[This corrects the article DOI: 10.1371/journal.pgen.1005485.].

Evaluation of intraductal delivery of poly(ethylene glycol)-doxorubicin conjugate nanocarriers for the treatment of ductal carcinoma in situ (DCIS)-like lesions in rats.

Ductal carcinoma in situ is the most commonly diagnosed early stage breast cancer. The efficacy of intraductally delivered poly(ethylene glycol)-doxorubicin (PEG-DOX) nanocarriers, composed of one or more DOX conjugated to various PEG polymers, was investigated in an orthotopic ductal carcinoma in situ-like rat model. In vitro cytotoxicity was evaluated against 13762 Mat B III cells using MTT assay. The orthotopic model was developed by inoculating cancer cells into mammary ducts of female Fischer 344 retired breeder rats. The ductal retention and in vivo antitumour efficacy of two of the six nanocarriers (5 kDa PEG-DOX and 40 kDa PEG-(DOX)4) were investigated based on in vitro results. Mammary retention of DOX and PEG-DOX nanocarriers was quantified using in vivo imaging. Histopathologic effects of DOX and PEG-DOX nanocarriers on mammary ductal structure were also investigated. Cytotoxicities of small linear PEG-DOX nanocarriers (5 and 10 kDa) were not different from DOX whereas larger PEG-DOX nanocarriers showed reduced potency. The order of mammary retention was 40 kDa PEG-(DOX)4 > 5 kDa PEG-DOX >> DOX, in normal and tumour-bearing rats. Intraductally administered PEG-DOX nanocarriers and DOX were effective in reducing tumour incidence and increasing survival rate, with no significant differences found among the three treatment groups. However, nanocarriers administered intravenously at the same doses were not effective, and intraductally administered free DOX caused severe local toxicity. Intraductal administration of PEG-DOX nanocarriers is effective and less toxic than that of free DOX, as well as IV DOX/PEG-DOX. Furthermore, PEG-DOX nanocarriers demonstrate the added benefit of prolonging DOX ductal retention, which would necessitate less frequent dosing.

The effect of size and polymer architecture of doxorubicin-poly(ethylene) glycol conjugate nanocarriers on breast duct retention, potency and toxicity.

Although systemic administration of chemotherapeutic agents is routinely used for treating invasive breast cancer, the only therapeutic options for ductal carcinoma in situ (DCIS) are surgery and radiation. Treating DCIS by delivering drugs locally to the affected milk duct offers significant advantages over systemic administration, including reduced systemic and breast toxicities, as well as a greatly reduced need for surgery and radiation. In this study, mammary gland retention and toxicity of intraductally administered poly(ethylene) glycol-doxorubicin (PEG-DOX) polymeric conjugate nanocarriers of varying molecular sizes and architectures were investigated. Nanocarriers were formed by conjugating one or more copies of doxorubicin to PEG polymers, of varying molecular weights (5, 10, 20, and 40 kDa) and architectures (linear, four-arm and eight-arm). Cytotoxicity against MCF7 cells, a human breast cancer cell line, was assessed, and IC50 values were calculated. The nanocarriers were intraductally administered into the mammary glands of female retired breeder Sprague-Dawley rats. Whole body images were captured using in vivo optical imaging, and changes in ductal structure as well local inflammation were monitored. Fluorescence intensities were monitored, over time, to evaluate nanocarrier mammary gland retention half-lives (t1/2). The IC50 values of PEG-DOX nanocarriers against MCF7 cells were 40 kDa PEG-(DOX)4 (1.23 µM) < 5 kDa PEG-DOX (1.76 µM) < 40 kDa PEG-(DOX)8 (3.49 µM) < 10 kDa PEG-DOX (3.86 µM) < 20 kDa PEG-DOX (8.96 µM) < 40 kDa PEG-DOX (18.11 µM), whereas the IC50 of free DOX was only 0.14 µM. The t1/2 of linear 5, 20, and 40 kDa nanocarriers were 2.2 ±â€¯0.3, 3.6 ±â€¯0.6, and 13.1 ±â€¯3.4 h, whereas the retention t1/2 of 4- and 8-arm 40 kDa nanocarriers were 14.9 ±â€¯5.6 h and 11.9 ±â€¯2.9 h, respectively. The retention t1/2 of free doxorubicin was 2.0 ±â€¯0.4 h, which was significantly shorter than that of the linear and branched 40 kDa PEG-DOX nanocarriers. Increased molecular weight and decreased branching both demonstrated a strong correlation to enhanced mammary gland retention. Intraductally administered free doxorubicin resulted in ductal damage, severe inflammation and generation of atypical cell neoplasms, whereas PEG-DOX nanocarriers induced only minor and transient inflammation (i.e., damaged epithelial cells and detached cellular debris). The 40 kDa 4-arm PEG-DOX nanocarrier demonstrated the longest ductal retention half-life, the lowest IC50 (i.e., most potent), and minimal ductal damage and inflammation. The current results suggest that PEG-DOX nanocarriers with prolonged ductal retention may present the best option for intraductal treatment of DCIS, due to their low local toxicity and potential for sustained therapeutic effect.

Preparation and properties of a novel covalently bonded energetic boron powder and its composite.

Promoting the ignition of boron powders in propellants, explosives and pyrotechnics has been a promising research direction. In this paper, a new strategy for covalently bonded energetic boron powders was designed. Specifically, 2,4-toluene diisocyanate (TDI) and 3-amino-1,2,4-triazole (ATZ) were used as grafting molecules, and then acidification, carbamation and ureylene addition were performed serially on the surface of the boron particles. The reaction conditions were optimized using infrared (IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). At lower temperatures, the addition of carbamation or ureylene can improve the yield and stability of isocyanate and urea groups. The chemical composition, microstructure and surface properties of the boron powders were analyzed with nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and contact angle (CA) analysis, respectively. The covalent bonding type was confirmed by the typical peaks at 155.7 and 157.5 ppm in the 13C NMR spectra of the intermediate isocyanate-grafting boron powders (TB) and target product triazole-grafting boron powders (TTB). The static water contact angles on the surfaces of TB and TTB were 148.3° and 37.0°, respectively. Influences of surface modification of boron powders on the rheological properties of boron/hydroxyl-terminated polybutadiene (HTPB) composites were investigated. Moreover, the prepared samples were characterized by thermogravimetry (TG) and differential scanning calorimetry (DSC) to investigate the thermal stability and reaction activity, and the results showed that this grafting strategy could significantly reduce the critical reaction temperature of B/KNO3. Consequently, it is anticipated that the modified boron powders can potentially be used in propellants, explosives and pyrotechnics with high impulses.

CCNYL1, but Not CCNY, Cooperates with CDK16 to Regulate Spermatogenesis in Mouse.

Cyclin Y-like 1 (Ccnyl1) is a newly-identified member of the cyclin family and is highly similar in protein sequences to Cyclin Y (Ccny). However, the function of Ccnyl1 is poorly characterized in any organism. Here we found that Ccnyl1 was most abundantly expressed in the testis of mice and was about seven times higher than the level of Ccny. Male Ccnyl1-/- mice were infertile, whereas both male and female Ccny-/- mice displayed normal fertility. These results suggest that Ccnyl1, but not Ccny, is indispensable for male fertility. Spermatozoa obtained from Ccnyl1-/- mice displayed significantly impaired motility, and represented a thinned annulus region and/or a bent head. We found that the protein, but not the mRNA, level of cyclin-dependent kinase 16 (CDK16) was decreased in the testis of Ccnyl1-/- mice. Further study demonstrated that CCNYL1 interacted with CDK16 and this interaction mutually increased the stability of these two proteins. Moreover, the interaction increased the kinase activity of CDK16. In addition, we observed an alteration of phosphorylation levels of CDK16 in the presence of CCNYL1. We identified the phosphorylation sites of CDK16 by mass spectrometry and revealed that several phosphorylation modifications on the N-terminal region of CDK16 were indispensable for the CCNYL1 binding and the modulation of CDK16 kinase activity. Our results therefore reveal a previously unrecognized role of CCNYL1 in regulating spermatogenesis through the interaction and modulation of CDK16.

Single-Step Assembly of Multimodal Imaging Nanocarriers: MRI and Long-Wavelength Fluorescence Imaging.

Magnetic resonance imaging (MRI)- and near-infrared (NIR)-active, multimodal composite nanocarriers (CNCs) are prepared using a simple one-step process, flash nanoprecipitation (FNP). The FNP process allows for the independent control of the hydrodynamic diameter, co-core excipient and NIR dye loading, and iron oxide-based nanocrystal (IONC) content of the CNCs. In the controlled precipitation process, 10 nm IONCs are encapsulated into poly(ethylene glycol) (PEG) stabilized CNCs to make biocompatible T2 contrast agents. By adjusting the formulation, CNC size is tuned between 80 and 360 nm. Holding the CNC size constant at an intensity weighted average diameter of 99 ± 3 nm (PDI width 28 nm), the particle relaxivity varies linearly with encapsulated IONC content ranging from 66 to 533 × 10(-3) m(-1) s(-1) for CNCs formulated with 4-16 wt% IONC. To demonstrate the use of CNCs as in vivo MRI contrast agents, CNCs are surface functionalized with liver-targeting hydroxyl groups. The CNCs enable the detection of 0.8 mm(3) non-small cell lung cancer metastases in mice livers via MRI. Incorporating the hydrophobic, NIR dye tris-(porphyrinato)zinc(II) into CNCs enables complementary visualization with long-wavelength fluorescence at 800 nm. In vivo imaging demonstrates the ability of CNCs to act both as MRI and fluorescent imaging agents.

A stationary north-finding scheme for an azimuth rotational IMU utilizing a linear state equality constraint.

The Kalman filter (KF) has always been used to improve north-finding performance under practical conditions. By analyzing the characteristics of the azimuth rotational inertial measurement unit (ARIMU) on a stationary base, a linear state equality constraint for the conventional KF used in the fine north-finding filtering phase is derived. Then, a constrained KF using the state equality constraint is proposed and studied in depth. Estimation behaviors of the concerned navigation errors when implementing the conventional KF scheme and the constrained KF scheme during stationary north-finding are investigated analytically by the stochastic observability approach, which can provide explicit formulations of the navigation errors with influencing variables. Finally, multiple practical experimental tests at a fixed position are done on a postulate system to compare the stationary north-finding performance of the two filtering schemes. In conclusion, this study has successfully extended the utilization of the stochastic observability approach for analytic descriptions of estimation behaviors of the concerned navigation errors, and the constrained KF scheme has demonstrated its superiority over the conventional KF scheme for ARIMU stationary north-finding both theoretically and practically.

Gelation chemistries for the encapsulation of nanoparticles in composite gel microparticles for lung imaging and drug delivery.

The formation of 10-40 µm composite gel microparticles (CGMPs) comprised of ∼100 nm drug containing nanoparticles (NPs) in a poly(ethylene glycol) (PEG) gel matrix is described. The CGMP particles enable targeting to the lung by filtration from the venous circulation. UV radical polymerization and Michael addition polymerization reactions are compared as approaches to form the PEG matrix. A fluorescent dye in the solid core of the NP was used to investigate the effect of reaction chemistry on the integrity of encapsulated species. When formed via UV radical polymerization, the fluorescence signal from the NPs indicated degradation of the encapsulated species by radical attack. The degradation decreased fluorescence by 90% over 15 min of UV exposure. When formed via Michael addition polymerization, the fluorescence was maintained. Emulsion processing using controlled shear stress enabled control of droplet size with narrow polydispersity. To allow for emulsion processing, the gelation rate was delayed by adjusting the solution pH. At a pH = 5.4, the gelation occurred at 3.5 h. The modulus of the gels was tuned over the range of 5 to 50 kPa by changing the polymer concentration between 20 and 70 vol %. NP aggregation during polymerization, driven by depletion forces, was controlled by the reaction kinetics. The ester bonds in the gel network enabled CGMP degradation. The gel modulus decreased by 50% over 27 days, followed by complete gel degradation after 55 days. This permits ultimate clearance of the CGMPs from the lungs. The demonstration of uniform delivery of 15.8 ± 2.6 µm CGMPs to the lungs of mice, with no deposition in other organs, is shown, and indicates the ability to concentrate therapeutics in the lung while avoiding off-target toxic exposure.

Core functional sequence of C-terminal GAG-binding domain directs cellular uptake of IGFBP-3-derived peptides.

The current study clarifies the role of the Glycosaminoglycan (GAG)-binding domain of insulin-like growth factor binding protein-3 (IGFBP-3) in cell penetration. The cell penetration function of IGFBP-3 has been mapped to an 18-residue GAG-binding domain in the C-terminal region that mobilizes cellular uptake and nuclear localization of unrelated proteins. Uptake of KW-22, a 22-residue peptide that encompasses the 18-residue GAG-binding domain, and another IGFBP-3 peptide carrying a streptavidin protein cargo was investigated in Chinese hamster ovary (CHO) cells defective at several steps of biosynthesis of cell surface GAGs. The severity of GAG truncation was highly correlated to the impairment of uptake ranging from complete abrogation to only a partial reduction, suggesting that GAG-binding is required for uptake. The 18-residue GAG-binding domain consists of an 8-residue KK-8 basic sequence devoid of Arg and an adjacent 10-residue QR-10 sequence rich in Arg. Peptide mapping of uptake and GAG-binding activities within the KW-22 peptide showed that the 8-residue KK-8 basic peptide retained 80% of GAG-binding activity with no uptake activity while the 10-residue QR-10 peptide retained 53% of uptake activity and 18% of GAG-binding activity. This suggests that KK-8 carries out the majority of GAG-binding function while QR-10 carries out the majority of the cell entry function. To our knowledge, this is the first report of physical separation of the uptake and GAG-binding functions within a short cell penetrating peptide and may shed light on the general mechanism of uptake of Arg-rich CPPs and guide new design of Arg-rich CPP-assisted drug/gene delivery systems.

Selective cytotoxicity and combined effects of camptothecin or paclitaxel with sodium-R-alpha lipoate on A549 human non-small cell lung cancer cells.

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer and remains the deadliest form of cancer in the United States and worldwide. New therapies are highly sought after to improve outcome. The effect of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity was evaluated on A549 NSCLC and BEAS-2B "normal" lung epithelial cells. Combination indices (CI) and dose reduction indices (DRI) were investigated by studying the cytotoxicity of sodium-R-alpha lipoate (0-16 mM), camptothecin (0-25 nM) and paclitaxel (0-0.06 nM) alone and in combination. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium-bromide (MTT) was used to assess cytotoxicity. The combinational cytotoxic effects of sodium-R-alpha lipoate with camptothecin or paclitaxel were analyzed using a simulation of dose effects (CompuSyn® 3.01). The effects of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity varied based on concentrations and treatment times. It was found that sodium-R-alpha lipoate wasn't cytotoxic toward BEAS-2B cells at any of the concentrations tested. For A549 cells, CIs [(additive (CI = 1); synergistic (CI < 1); antagonistic (CI < 1)] were lower and DRIs were higher for the camptothecin/sodium-R-alpha-lipoate combination (CI = ∼0.17-1.5; DRI = ∼2.2-22.6) than the paclitaxel/sodium-R-alpha-lipoate combination (CI = ∼0.8-9.9; DRI = ∼0.10-5.8) suggesting that the camptothecin regimen was synergistic and that the addition of sodium-R-alpha lipoate was important for reducing the camptothecin dose and potential for adverse effects.

Biodistribution and renal clearance of biocompatible lung targeted poly(ethylene glycol) (PEG) nanogel aggregates.

A novel stabilized aggregated nanogel particle (SANP) drug delivery system was prepared for injectable passive lung targeting. Gel nanoparticles (GNPs) were synthesized by irreversibly cross-linking 8 Arm PEG thiol with 1,6-hexane-bis-vinylsulfone (HBVS) in phosphate buffer (PB, pH 7.4) containing 0.1% v/v Tween™ 80. Aggregated nanogel particles (ANPs) were generated by aggregating GNPs to micron-size, which were then stabilized (i.e., SANPs) using a PEG thiol polymer to prevent further growth-aggregation. The size of SANPs, ANPs and GNPs was analyzed using a Coulter counter and transmission electron microscopy (TEM). Stability studies of SANPs were performed at 37°C in rat plasma, phosphate buffered saline (PBS, pH 7.4) and PB (pH 7.4). SANPs were stable in rat plasma, PBS and PB over 7 days. SANPs were covalently labeled with HiLyte Fluor™ 750 (DYE-SANPs) to facilitate ex vivo imaging. Biodistribution of intravenous DYE-SANPs (30 µm, 4 mg in 500 µL PBS) in male Sprague-Dawley rats was compared to free HiLyte Fluor™ 750 DYE alone (1mg in 500 µL PBS) and determined using a Xenogen IVIS® 100 Imaging System. Biodistribution studies demonstrated that free DYE was rapidly eliminated from the body by renal filtration, whereas DYE-SANPs accumulated in the lung within 30 min and persisted for 48 h. DYE-SANPs were enzymatically degraded to their original principle components (i.e., DYE-PEG-thiol and PEG-VS polymer) and were then eliminated from the body by renal filtration. Histological evaluation using H & E staining and broncho alveolar lavage (BAL) confirmed that these flexible SANPs were not toxic. This suggests that because of their flexible and non-toxic nature, SANPs may be a useful alternative for treating pulmonary diseases such as asthma, pneumonia, tuberculosis and disseminated lung cancer.

Influence of molecular size on the retention of polymeric nanocarrier diagnostic agents in breast ducts.

PURPOSE: To investigate the influence of nanocarrier molecular size and shape on breast duct retention in normal rats using a non-invasive optical imaging method. METHODS: Fluorescein-labeled PEG nanocarriers of different molecular weights and shapes (linear, two-arm, four-arm, and eight-arm) were intraductally administered (50 nmol) to female Sprague-Dawley rats. Whole body images were obtained non-invasively. Fluorescence intensities (i.e., amount remaining in duct) were plotted against time to estimate the nanocarrier ductal retention half-lives (t(1/2)). Plasma samples were taken and the pharmacokinetics (Tmax, Cmax) of absorbed nanocarriers was also assessed. RESULTS: The t(1/2) of linear 12, 20, 30, 40, and two-arm 60 kDa nanocarriers were 6.7 ± 0.9, 16.1 ± 4.1, 16.6 ± 3.4, 21.5 ± 2.7, and 19.5 ± 6.1 h, whereas the four-arm 20, 40, and eight-arm 20 kDa had t(1/2) of 9.0 ± 0.5, 11.5 ± 1.9, and 12.6 ± 3.0 h. The t(1/2) of unconjugated fluorescein was significantly lower (14.5 ± 1.4 min). The Tmax for 12, 40, 60 kDa nanocarriers were 1, 24, and 32 h, respectively, and only 30 min for fluorescein. CONCLUSIONS: Since normal breast ducts are highly permeable, the use of nanocarriers may be helpful in prolonging ductal retention of diagnostic and/or therapeutic agents.

Noninvasive detection of passively targeted poly(ethylene glycol) nanocarriers in tumors.

The present studies noninvasively investigate the passive tumor distribution potential of a series of poly(ethylene glycol) (PEG) nanocarriers using a SkinSkan spectrofluorometer and an In Vivo Imaging System (IVIS) 100. Fluorescein conjugated PEG nanocarriers of varying molecular weights (10, 20, 30, 40, and 60 kDa) were prepared and characterized. The nanocarriers were administered intravenously to female balb/c mice bearing subcutaneous 4T1 tumors. Passive distribution was measured in vivo (λ(exc), 480 nm; λ(em), 515-520 nm) from the tumor and a contralateral skin site (i.e., control site). The signal intensity from the tumor was always significantly higher than that from the contralateral site. Trends in results between the two methods were consistent with tumor distribution increasing in a molecular weight-dependent manner (10 < 20 < 30 ≪ 40 ≪ 60 kDa). The 10 kDa nanocarrier was not detected in tumors at 24 h, whereas 40-60 kDa nanocarriers were detected in tumors for up to 96 h. The 30, 40, and 60 kDa nanocarriers showed 2.1, 5.3, and 4.1 times higher passive distribution in tumors at 24 h, respectively, as compared to the 20 kDa nanocarrier. The 60 kDa nanocarrier exhibited 1.5 times higher tumor distribution than 40 kDa nanocarrier at 96 h. Thus, PEG nanocarriers (40 and 60 kDa) with molecular weights close to or above the renal exclusion limit, which for globular proteins is ≥45 kDa, showed significantly higher tumor distribution than those below it. The hydrodynamic radii of PEG polymers, measured using dynamic light scattering (DLS), showed that nanocarriers obtained from polymers with hydrodynamic radii ≥8 nm exhibited higher tumor distribution. Ex vivo mass balance studies revealed that nanocarrier tissue distribution followed the rank order tumor > lung > spleen > liver > kidney > muscle > heart, thus validating the in vivo studies. The results of the current studies suggest that noninvasive dermal imaging of tumors provides a reliable and rapid method for the initial screening of nanocarrier tumor distribution pharmacokinetics.

Biodegradable poly(ethylene glycol) hydrogels based on a self-elimination degradation mechanism.

Two vinyl sulfone functionalized crosslinkers were developed for the purpose of preparing degradable poly(ethylene glycol) (PEG) hydrogels (EMXL and GABA-EMXL hydrogels). A self-elimination degradation mechanism in which an N-terminal residue of a glutamine is converted to pyroglutamic acid with subsequent release of diamino PEG (DAP) is proposed. The hydrogels were formed via Michael addition by mixing degradable or nondegradable crosslinkers and copolymer {4% w/v; poly[PEG-alt-poly(mercapto-succinic acid)]} at room temperature in phosphate buffer (PB, pH = 7.4). Hydrogel degradation was characterized by assessing diamino PEG release and examining morphological changes as well as the swelling and weight loss ratio under physiological conditions (37 degrees C). Degradation of EMXL and GABA-EMXL hydrogels occurred by surface erosion (confirmed by SEM). GABA-EMXL degradation was significantly faster (approximately 3-fold) than EMXL; however, the degradation of both hydrogels in mouse plasma was 12-times slower than in PBS. The slower degradation rate in plasma as compared to buffer is consistent with the presence of gamma-glutamyltransferase, gamma-glutamylcyclotransferase and/or glutaminyl cyclase (QC), which have been shown to suppress pyroglutamic acid formation. The current studies suggest that EMXL and GABA-EMXL hydrogels may have biomedical applications where 1-2 week degradation timeframes are optimal.