HPMA Copolymer-Anticancer Drug-OV-TL-TL16 Antibody Conjugates. 1. Influence of the Method of Synthesis on the Biding Affinity to OVCAR-3 Ovarian Carcinoma Cells In Vitro

The influence of different methods of binding the OV-TL16 antibody and its Fab′ fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer—drug (adriamycin {ADR} or meso chlorin e₆ mono(N-2-aminoethylamide) {Mce₆} conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer—drug (ADR or Mce₆) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling the HPMA copolymer—Mce₆ conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab′ fragment of the OV-TL16 antibody demonstrated a more homogeneous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physicochemical methods where employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce₆) and the spectral properties of Mce₆ were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with changes in antibody conformation.     

Do HPMA copolymer conjugates have a future as clinically useful nanomedicines? A critical overview of current status and future opportunities

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing doxorubicin designed in the late 1970s/early 1980s as anticancer polymer therapeutics were the first synthetic polymer-based anticancer conjugates to enter clinical trial beginning in 1994. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA copolymers as a new polymer platform in this setting. Subsequent Phase I/II trials have investigated conjugates containing paclitaxel (PNU 166945), camptothecin (PNU 166148) (both failed in clinical trials underlining the importance of rational design), and most recently HPMA-copolymer platinates (AP5280 and then AP5346-ProLindac^TM) entered Phase II clinical development. There are a growing array of second generation HPMA copolymer-based systems involving combination therapy, incorporating putative targeting ligands, having an ever more complex architecture, and both drug and protein conjugates are being proposed as novel treatments for diseases other than cancer. Despite their promise, and the success of polymeric drugs and polymer-protein conjugates, no polymer-drug conjugate (HPMA copolymer-based or otherwise) has yet entered routine clinical use. It is timely to reflect on the progress made over the last 30 years, the relative merits of HPMA copolymers as a platform compared to other polymeric carriers, and comment on their future potential as polymer-based nanomedicines into the 21st century in comparison with the many alternative strategies now emerging for creation of nanopharmaceuticals.     

HPMA Copolymer-Aminohexylgeldanamycin Conjugates Targeting Cell Surface Expressed GRP78 in Prostate Cancer

Purpose  

This study focused on the synthesis and in vitro characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for the delivery of geldanamycin to prostate cancer tumors. Conjugates were modified to incorporate WIFPWIQL peptide, which binds to cell-surface-expressed Glucose-regulated protein 78.     

Synthesis and in vitro anti-tumor activity of novel HPMA copolymer-drug conjugates with potential cell surface targeting property for carcinoma cells

In several groups of malignant tumors including head and neck tumors, a protein named Hsp47/CBP2 leaked from the cell was expressed on the tumor cell surface. Several synthetic peptides have been identified as effective ligands for binding to Hsp47/CBP2. This study has focused on the synthesis and in vitro characterization of a targeting delivery system of 5-fluorouracil (5-FU) to human head and neck squamous cell carcinoma (HNSCC) in order to improve anti-cancer efficacy and reduce dose-limiting toxicity of 5-FU. An N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, with Hsp47/CBP2 binding peptide sequence (namely WHYPWFQNWAMA) as a targeting ligand, was synthesized by a novel and simplified synthetic route. Under the controlled synthetic conditions, 1,3-dimethylol-5-FU, derived from 5-FU, was attached to the HPMA copolymer backbone via the lysosomally degradable GFLG linker, while the WHYPWFQNWAMA was conjugated via a non-degradable Gly-Gly (GG) linker. A control polymer without targeting moiety was also synthesized (P-FU). The in vitro cytotoxicity, internalization and apoptosis assays of the polymeric conjugates were evaluated. The characteristic apoptotic morphological changes were also assessed. Compared to 5-FU and P-FU, the HPMA copolymer containing the Hsp47/CBP2 binding peptide (P-FU-peptide) exhibited the highest cytotoxic efficacy to cell line of human head and neck squamous cell carcinoma (p < 0.05) and was internalized much faster than P-FU, especially after being incubated for 30 min. Both of the morphology and apoptosis analyses demonstrated that the treatment of P-FU-peptide resulted in more apoptotic and necrotic induction of tumor cells than P-FU. Meanwhile, the rate of apoptosis induced by P-FU-peptide was higher than that of necrosis. In summary, the HPMA copolymer-Hsp47/CBP2 binding peptide conjugates showed a promising future for the treatment of HNSCC with improved efficacy.     

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method

The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10⁻⁶ cm/s) across the Caco-2 cell monolayer, and the ratio of P_A–B to P_B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences (P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P_w = 6.18), followed by colon (P_w = 2.07), duodenum (P_w = 0.61) and jejunum (P_w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.     

In vitro human plasma distribution of nanoparticulate paclitaxel is dependent on the physicochemical properties of poly(ethylene glycol)-block-poly(caprolactone) nanoparticles

In this study, we synthesized and characterized two methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-b-PCL) amphiphilic diblock copolymers, both based on MePEG with a molecular weight of 5000 g/mol (114 repeat units) and PCL block lengths of either 19 or 104 repeat units. Nanoparticles were formed from these copolymers by a nanoprecipitation and dialysis technique. The MePEG₁₁₄-b-PCL₁₉ copolymer was water soluble and formed micelles that had a hydrodynamic diameter of 40 nm at all copolymer concentrations tested, and displayed a relatively low core microviscosity. The practically water insoluble MePEG₁₁₄-b-PCL₁₀₄ copolymer formed nanoparticles with a larger hydrodynamic diameter, which was dependent on copolymer concentration, and possessed a higher core microviscosity than the MePEG₁₁₄-b-PCL₁₉ micelles, characteristic of nanospheres. The micelles solubilized a maximum of 1.6% w/w of the hydrophobic anticancer agent, paclitaxel (PTX), and released 92% of their drug payload over 7 days, as compared to the nanospheres, which solubilized a maximum of 3% w/w of PTX and released 60% over the same period of time. Both types of nanoparticles were found to be hemocompatible, causing only minimal hemolysis and no changes in plasma coagulation times as compared to control. Upon in vitro incubation in human plasma, PTX solubilized by micelles had a plasma distribution similar to free drug. The majority of PTX was associated with the lipoprotein deficient plasma (LPDP) fraction, which primarily consists of albumin and alpha-1-acid glycoprotein. In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction.     

正电化修饰的HPMA聚合物-阿霉素接合物的制备表征及对肿瘤细胞摄取的影响

目的:制备2种正电化修饰的N-(2-羟丙基)甲基丙烯酰胺(HPMA)聚合物-阿霉素接合物并表征,分别考察2种接合物的正电基团含量对肿瘤细胞摄取的影响。方法:制备侧链带伯胺基的HPMA聚合物-阿霉素接合物(pHPMA-DOX-APMA)和侧链带胍基的HPMA聚合物-阿霉素接合物(pHPMA-DOX-GPMA),对其药剂学性质如正电基团含量,载药量,Zeta电位和分子量进行表征,进一步考察不同正电基团含量的接合物对MCF-7细胞摄取和毒性的影响。结果:通过自由基聚合反应,2种接合物成功合成。其中pHPMA-DOX-APMA伯胺基含量为0.44~1.57 mmol·g^-1,载药量为7.15%~9.25%;pHPMA-DOX-GPMA胍基含量为0.11~0.54 mmol·g^-1,载药量为7.55%~9.07%;相对分子质量分别为33~38 kDa和32~37 kDa。通过BCA法和MTT法研究分别发现在pHPMA-DOX-APMA中的伯胺基团含量为1.570 mmol·g^-1及pHPMA-DOX-GPMA中的胍基含量为0.260 mmol·g^-1时,肿瘤细胞对阿霉素的摄取量显著增加,二者的IC₅₀与pHPMA-DOX相比显著降低(P<0.05)。结论:成功制备了2种正电化修饰的HPMA聚合物-阿霉素接合物;适当的正电化修饰对阿霉素的肿瘤细胞摄取有促进作用。     

Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates

The biodistribution and pharmacokinetics of bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-alendronate conjugates were evaluated following intravenous administration of radioiodinated conjugates to young healthy BALB/c mice. The synthesis of a polymerizable and cathepsin K cleavable alendronate derivative, N-methacryloylglycylglycylprolylnorleucylalendronate, enabled the preparation of HPMA copolymer-alendronate conjugates with varying composition. Using the RAFT (reversible addition-fragmentation chain transfer) polymerization technique, four conjugates with different molecular weight and alendronate content and two control HPMA copolymers (without alendronate) with different molecular weight were prepared. The results of biodistribution studies in mice demonstrated a strong binding capacity of alendronate-targeted HPMA copolymer conjugates to bone. Conjugates with low (1.5 mol%) alendronate content exhibited a similar bone deposition capacity as conjugates containing 8.5 mol % of alendronate. The molecular weight was an important factor in the biodistribution of the HPMA copolymer conjugates. More conjugate structures need to be evaluated, but the data suggest that medium molecular weights (50-100 kDa) might be effective drug carriers for bone delivery.     

Evaluation of long-circulating nanoparticles using biodegradable ABA triblock copolymers containing of poly(l-lactic acid) A-blocks attached to central poly(oxyethylene) B-blocks in vivo

The nanoparticles (mean diameter 152–377 nm) consisting of the ABA triblock copolymers (M_w 29 000–147 000) containing poly(l-lactic acid) (PLA) A-blocks attached to central poly(oxyethylene) (PEG) B-blocks (PEG M_w 6600, 20 000) (PLA-PEG-PLA) were prepared, and the effects of the polymer characteristics on the pharmacokinetics of the nanoparticles and the biodistribution of the nanoparticles were studied. Progesterone was used as a model drug. We could make the long-circulating nanoparticles using the triblock copolymer. The degree of burst in early phase, which was estimated by the Co values (Progesterone concentration at time 0) and the circulating-time were affected by the total M_w, the PEG content, the PEG M_w and the M_w/M_n (M_n: where M_n is the number average molecular weight) ratio. The purification of the polymer was an important factor for the control of the burst. These triblock copolymer nanoparticles induced reduction in the liver and spleen uptake of the nonoparticles. These phenomena are probably explained by the avoidence of adsorption of opsonin to the particles as a result of the orientation of PEG on the surface of the particles.     

Influence of the Structure of Drug Moieties on the in Vitro Efficacy of HPMA Copolymer–Geldanamycin Derivative Conjugates

Purpose To optimize the structure of geldanamycin (GDM) derivative moieties attached to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via an enzymatically degradable spacer. Methods HPMA copolymers containing different AR-GDM (AR = 3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxy-propyl AP(OH)) were synthesized and characterized. Their cytotoxicity towards the A2780 human ovarian carcinoma cells was evaluated. Results The cytotoxic efficacy of HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM. Particularly, HPMA copolymer-bound AH-GDM, which possessed the longest substituent at the 17-position, demonstrated the highest efficacy among the polymer-bound GDM derivatives; however the activity of free AH-GDM was lower than that of the other free AR-GDMs. The relative increase of the activity of macromolecular AH-GDM when compared to AP-GDM or AP(OH)-GDM correlated with the enhanced recognition of AH-GDM terminated oligopeptide side-chains by the active site of the lysosomal enzyme, cathepsin B Drug stability and further stabilization upon binding to HPMA copolymer also contributed to the observed phenomena. Conclusion AH-GDM was found to be a suitable GDM derivative for the design of a drug delivery system based on HPMA copolymers and enzymatically-degradable spacers.     

Overcoming the stromal barrier for targeted delivery of HPMA copolymers to pancreatic tumors

Delivery of macromolecules to pancreatic cancer is inhibited by a dense extracellular matrix composed of hyaluronic acid, smooth muscle actin and collagen fibers. Hyaluronic acid causes a high intratumoral fluidic pressure which prevents diffusion and penetration into the pancreatic tumor. This study involves the breaking down of hyaluronic acid by treating CAPAN-1 xenograft tumors in athymic nu/nu mice with targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers radiolabeled with ¹¹¹In for single photon emission computerized tomography (SPECT) imaging. Two targeting strategies were investigated including α_vβ₃ integrin and HER2 receptors. HPMA copolymers were targeted to these receptors by conjugating short peptide ligands cRGDfK and KCCYSL to the side chains of the copolymer. Results demonstrate that tumor targeting can be achieved in vivo after treatment with hyaluronidase. This approach shows promise for enhanced delivery of polymer–peptide conjugates to solid tumors.     

Targetable HPMA Copolymer–Aminohexylgeldanamycin Conjugates for Prostate Cancer Therapy

Purpose  This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. Materials and Methods  HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to α_vβ₃ integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. Results  HPMA copolymers containing AH-GDM and RGDfK showed active binding to the α_vβ₃ integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer–AH-GDM–RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. Conclusion  Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer–AH-GDM–RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.     

Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments

Hyaluronan (HA) has been extensively used for various medical applications, including osteoarthritis, tissue augmentation and ocular surgery. More recently, it has been investigated for use in polymer therapeutics as a carrier for drugs and biologically active proteins, thanks to its biodegradability, biocompatibility and inherent biological properties. Such biological functions are strongly dependent on HA's chain length, yet the molecular weight of HAs used in polymer conjugates varies widely and is inconsistent with its intended application. Therefore, this study aimed to determine the ideal chain length of HA to be used in polymer conjugates for enhanced tissue repair.HA fragments (M_w 45,000-900,000 g/mol) were prepared by acid hydrolysis of rooster comb HA and their physicochemical and biological properties were characterized. Such HA fragments had a highly extended, almost rod-like solution conformation and demonstrated chain length- and concentration-dependent viscosity, while exposure to HAase caused a rapid reduction in HA viscosity, which was most significant for the native HA. Initial HA hydrolysis rate by HAase varied strongly with HA chain length and was dependent on the formation of a stable enzyme-substrate complex. When normal human dermal fibroblasts were exposed to the different HA fragments for 72 h, only native (900,000 g/mol) HA reduced proliferation at 1000 μg/mL. Conversely, only the smallest HA fragment (70,000 g/mol) reduced the proliferation of chronic wound fibroblasts, at 1000 μg/mL. The 70,000 g/mol HA fragment also promoted the greatest cell attachment.These observations demonstrate that low molecular weight (70,000-120,000 g/mol) HA fragments would be best suited for the delivery of proteins and peptides with applications in chronic wound healing and paves the way for the rationalized development of novel HA conjugates.     

Water-soluble HPMA copolymer—prostaglandin E1 conjugates containing a cathepsin K sensitive spacer

A novel bone targeting, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based, prostaglandin E₁ (PGE₁) delivery system was designed, synthesized and characterized. PGE₁ was bound to the polymer backbone via a spacer, composed of a cathepsin K sensitive tetrapeptide (Gly-Gly-Pro-Nle) and a self-eliminating 4-aminobenzyl alcohol structure. The HPMA copolymer conjugates were prepared by photo–initiated free radical copolymerization of HPMA, PGE₁-containing macromonomer, and optionally a comonomer containing a reactive p-nitrophenyl ester group. The latter group was used as attachment points for the d-aspartic acid octapeptide targeting moieties. Incubation of the PGE₁-containing macromonomer and HPMA copolymer-PGE₁ conjugates with cathepsin K resulted in release of unmodified PGE₁. The rate of release depended on the composition of the conjugate. The higher the PGE₁ content in the conjugate, the slower the PGE₁ release. This appeared to be the result of association of hydrophobic side-chains in aqueous media, which rendered the formation of the enzyme substrate complex more difficult. The data seems to indicate that HPMA copolymer-PGE₁ conjugates have a potential in the treatment of osteoporosis and other bone diseases.     

HPMA Copolymer-1,5-Diazaanthraquinone Conjugates as Novel Anticancer Therapeutics

1,5-Diazaanthraquinones (DAQs) are promising anticancer drugs, however, their clinical potential is limited due to poor solubility. Conjugation of anticancer agents to hydrophilic water-soluble polymers can overcome this problem and has already been used to generate conjugates with demonstrated clinical benefit. Here a library of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing a novel amino-functionalised 1,5-diazaanthraquinone derivative (amino-DAQ) have been synthesised. The conjugates were fully characterised by UV, HPLC, SEC, FT-Raman and NMR spectroscopy. Conjugation to HPMA copolymers improved amino-DAQ aqueous solubility (>7-fold). The HPMA copolymer-amino-DAQ conjugates were slightly less haemolytic than the parent compound (2% Hb released in 1 h for conjugate HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate compared to 13% obtained with amino-DAQ). When conjugates were incubated with isolated rat liver lysosomal enzymes (Tritosomes) the rate of amino-DAQ release was influenced by both drug loading and the composition of the peptidyl side chain used to link the drug to the carrier. The higher the drug loading the lower the rate of drug release. Whereas the GG linker did not release amino-DAQ, up to 26% of the amino-DAQ was released from a GFLG linker over 24 h. The in vitro cytotoxicity of these conjugates was evaluated against two different cell lines, B16F10 murine melanoma and MCF-7 human breast cancer cells. HPMA copolymer-amino-DAQ conjugates, which are internalised by cells by the endocytic pathway, showed much lower in vitro cytotoxicity (IC₅₀ for HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate > 397 μM drug-equiv.) than the free drug (the IC₅₀ for amino-DAQ was 12.6 and 2.8 μM against the B16F10 murine melanoma and the MCF-7 breast cancer cell line, respectively). Nonetheless, the observed lysosomal activation of the HPMA copolymer-GFLG-amino-DAQ conjugates, suggests that evaluation of the antitumour potential in vivo is warranted.     

Leptin and IL-8: Two novel cytokines screened out in childhood lead exposure

Lead is a toxic heavy metal with many recognized adverse health side effects. The central nervous system is the main target of lead toxicity. Although many studies on lead toxicity were conducted, the mechanism of lead toxicity remains uncertain. One possible attribution is the immature blood–brain barrier that causes lead exposure in children. Few studies have investigated the cytokine changes caused by this exposure. Novel cytokines were detected by RayBio^® Human Cytokine Antibody Array and validated by enzyme-linked immunosorbent assay. Several children were admitted to West China Second University Hospital, after a serious lead pollution event in longchang, Sichuan, China. A total of 4 children with elevated blood lead levels (BLLs) and 4 children with low BLLs were randomly chosen in the discovery set, and 40 children with elevated BLLs and 40 children with low BLLs were included in the validation set. Leptin and interleukin-8 (IL-8) were identified to be significantly different between children with elevated and low BLLs via RayBio^® Human Cytokine Antibody Array. In the validation set, IL-8 was higher in children with elevated BLLs [median(P₂₅–P₇₅), 117.69(52.31–233.63) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 17.70(10.75–26.52) pg/mL] (p = 0.000). Leptin was lower in children with elevated BLLs [median(P₂₅–P₇₅): 1658.23(1421.86–2606.55) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 4168.68(3246.32–4744.94) pg/mL] (p = 0.000). In children with low BLLs, leptin was higher in children with BLLs < 3 μg/dL (N = 7) [median(P25–P75): 7220.86(4265.72–7555.15) pg/mL] than in children with BLL ≥ 3 μg/dL (N = 33) [median(P₂₅–P₇₅): 4103.86(3163.40–4678.34) pg/mL] (p = 0.026); IL-8 was significantly different in children with BLL < 4 μg/dL (N = 13) [median(P₂₅–P₇₅): 12.49(8.25–14.86) pg/mL] than in children with BLL ≥ 4 μg/dL (N = 27) [median(P₂₅–P₇₅): 21.98(13.64–33.50) pg/mL] (p = 0.013). The results defined specific changes in cytokine expressions to lead exposure, which can be used to explore the mechanism of lead toxicity and monitor lead exposure.     

Noninvasive Visualization of Pharmacokinetics,Biodistribution and Tumor Targeting of Poly[<Emphasis Type="BoldItalic">N</Emphasis>-(2-hydroxypropyl)methacrylamide] in Mice Using Contrast Enhanced MRI

Purpose To study a non-invasive method of using contrast enhanced magnetic resonance imaging (MRI) to visualize the real-time pharmacokinetics, biodistribution and tumor accumulation of paramagnetically labeled poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) copolymer conjugates with different molecular weights and spacers in tumor-bearing mice. Materials and Methods Paramagnetically labeled HPMA copolymer conjugates were synthesized by free radical copolymerization of HPMA with monomers containing a chelating ligand, followed by complexation with Gd(OAc)₃. A stable paramagnetic chelate, Gd-DO3A, was conjugated to the copolymers via a degradable spacer GlyPheLeuGly and a non-degradable spacer GlyGly, respectively. The conjugates with molecular weights of 28, 60 and 121 kDa and narrow molecular weight distributions were prepared by fractionation with size exclusion chromatography. The conjugates were injected into athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts via a tail vein. MR images were acquired before and at various time points after the injection with a 3D FLASH sequence and a 2D spin-echo sequence at 3T. Pharmacokinetics, biodistribution and tumor accumulation of the conjugates were visualized based on the contrast enhancement in the blood, major organs and tumor tissue at various time points. The size effect of the conjugates was analyzed among the conjugates. Results Contrast enhanced MRI resulted in a real-time, three-dimensional visualization of blood circulation, pharmacokinetics, biodistribution and tumor accumulation of the conjugates, and the size effect on these pharmaceutical properties. HPMA copolymer conjugates with high molecular weight had a prolonged blood circulation time and high passive tumor targeting efficiency. Non-biodegradable HPMA copolymers with molecular weights higher than the threshold of renal filtration demonstrated higher efficiency for tumor drug delivery than biodegradable poly(L-glutamic acid). Conclusions Contrast enhanced MRI is an effective method for non-invasive visualization of in vivo properties of the paramagnetically labeled polymer conjugates in preclinical studies.     

Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro

Although some types of carbon nanotubes (CNTs) have been described to induce mesothelioma in rodents and genotoxic effects in various cell systems, there are few previous studies on the genotoxicity of CNTs in mesothelial cells. Here, we examined in vitro DNA damage induction by short multi-wall CNTs (MWCNTs; 10–30 nm × 1–2 μm) and single-wall CNTs (SWCNTs; >50% SWCNTs, ∼40% other CNTs; <2 nm × 1–5 μm) in human mesothelial (MeT-5A) cells and bronchial epithelial (BEAS 2B) cells, using the single cell gel electrophoresis (comet) assay and the immunoslot blot assay for the detection of malondialdehyde (M₁dG) DNA adducts. In BEAS 2B cells, we also studied the induction of micronuclei (MN) by the CNTs using the cytokinesis-block method. The cells were exposed to the CNTs (5–200 μg/cm², corresponding to 19–760 μg/ml) for 24 and 48 h in the comet assay and for 48 and 72 h in the MN and M₁dG assays. Transmission electron microscopy (TEM) showed more MWCNT fibres and SWCNT clusters in BEAS 2B than MeT-5A cells, but no significant differences were seen in intracellular dose expressed as area of SWCNT clusters between TEM sections of the cell lines. In MeT-5A cells, both CNTs caused a dose-dependent induction of DNA damage (% DNA in comet tail) in the 48-h treatment and SWCNTs additionally in the 24-h treatment, with a statistically significant increase at 40 μg/cm² of SWCNTs and (after 48 h) 80 μg/cm² of both CNTs. SWCNTs also elevated the level of M₁dG DNA adducts at 1, 5, 10 and 40 μg/cm² after the 48-h treatment, but both CNTs decreased M₁dG adduct level at several doses after the 72-h treatment. In BEAS 2B cells, SWCNTs induced a statistically significant increase in DNA damage at 80 and 120 μg/cm² after the 24-h treatment and in M₁dG adduct level at 5 μg/cm² after 48 h and 10 and 40 μg/cm² after 72 h; MWCNTs did not affect the level of DNA damage but produced a decrease in M₁dG adducts in the 72-h treatment. The CNTs did not affect the level of MN. In conclusion, MWCNTs and SWCNTs induced DNA damage in MeT-5A cells but showed a lower (SWCNTs) or no (MWCNTs) effect in BEAS 2B cells, suggesting that MeT-5A cells were more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, despite the fact that more CNT fibres or clusters were seen in BEAS 2B than MeT-5A cells. M1dG DNA adducts were induced by SWCNTs but decreased after a 3-day exposure to MWCNTs and (in MeT-5A cells) SWCNTs, indicating that CNTs may lead to alterations in oxidative effects within the cells. Neither of the CNTs was able to produce chromosomal damage (MN).     

Binding and Cytotoxicity of HPMA Copolymer Conjugates to Lymphocytes Mediated by Receptor-Binding Epitopes

Purpose. Studies on the recognition of epitopes presented on a template peptide showed the potential of nonapeptide-related sequences to act as biorecognition sites for the B-cell CD21 receptor. This study was intended to evaluate the capability of three epitope sequences to mediate specific cell binding and to enhance the cytotoxicity of HPMA copolymer conjugates. Methods. HPMA copolymer conjugates were synthesized containing three different epitopes at various contents and either a fluorescent marker or doxorubicin (DOX). The binding and cytotoxicity of the conjugates to CD21⁺ Raji B cells and CD21^– HSB-2 T cells were evaluated. Results. The epitope-containing conjugates were found to bind to Raji cells at different apparent affinities depending on epitope structure and content. The conjugates generally possessed higher affinities for Raji cells than for HSB-2 cells. Targeted HPMA copolymer-DOX conjugates exhibited higher cytotoxicities than the nontargeted conjugate, likely indicative of enhanced internalization by receptor-mediated endocytosis. HSB-2 cells were more sensitive to both free and polymer-bound DOX than Raji cells; however, the enhancement of cytotoxicity of the conjugates by incorporation of epitopes was more pronounced for Raji cells. Conclusions. The results verified the concept of using receptor-binding epitopes as targeting moieties in HPMA copolymer conjugates for the delivery of anticancer drugs to lymphoma cells.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.