Saijie Zhu

Partly PEGylated polyamidoamine dendrimer for tumor-selective targeting of doxorubicin: The effects of PEGylation degree and drug conjugation style

Partly PEGylated polyamidoamine (PAMAM) dendrimers were used as the carrier for tumor-selective targeting of the anticancer drug doxorubicin (DOX). Acid-sensitive cis-aconityl linkage or acid-insensitive succinic linkage was introduced between DOX and polymeric carriers to produce PPCD or PPSD conjugates, respectively. DOX release from PPCD conjugates followed an acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PPCD conjugates against murine B16 melanoma cells increased with, while cellular uptake decreased with increasing PEGylation degree. PPSD conjugates released negligible drug at any tested pH condition and were less cytotoxic. Confocal laser scanning microscopy confirmed the acid-sensitive release of DOX from PPCD conjugates in the lysosomes and the entrance into nuclei. Pharmacokinetic and biodistribution studies demonstrated that increasing PEGylation degree resulted in reduced liver and splenic accumulation, longer circulation time and more tumor accumulation of the conjugates. Although PPSD conjugates showed more tumor accumulation than PPCD conjugates at the same PEGylation degree, the acid-sensitive DOX release from PPCD conjugates ensured higher concentration of free DOX in tumor and more pronounced antitumor activity. Besides, the antitumor activity of PPCD conjugates increased with increasing PEGylation degree. Overall, PPCD conjugate with the highest PEGylation would be a promising candidate for solid tumor therapy.

PEGylated PAMAM Dendrimer-Doxorubicin Conjugates: In Vitro Evaluation and In Vivo Tumor Accumulation

PurposeTo investigate the effects of PEGylation degree and drug conjugation style on the in vitro and in vivo behavior of PEGylated polyamidoamine (PAMAM) dendrimers-based drug delivery system.MethodsDoxorubicin (DOX) was conjugated to differently PEGylated PAMAM dendrimers by acid-sensitive cis-aconityl linkage and acid-insensitive succinic linkage to produce the products of PPCD and PPSD conjugates, respectively. In vitro evaluations including pH-dependent DOX release, cytotoxicity, cellular uptake, cell internalization mechanism, and intracellular localization were performed. Tumor accumulation was also visualized by in vivo fluorescence imaging.ResultsDOX release from PPCD conjugates followed an acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PPCD conjugates against ovarian cancer (SKOV-3) cells increased, while cellular uptake decreased with increasing PEGylation degree. PPSD conjugates released negligible drug at any tested pH condition and were less cytotoxic. The conjugates were internalized by SKOV-3 cells via clathrin-mediated and adsorptive endocytosis, and were delivered to acidic lysosomes where DOX was released from PPCD conjugates and diffused into the nuclei. PPCD conjugates with highest PEGylation degree showed the highest tumor accumulation in mice inoculated with SKOV-3 cells.ConclusionThe obtained results suggested that PPCD conjugates with highest PEGylation degree would be a potential candidate for solid tumor treatment.

Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin.

The aim of the present report was to exploit the possibility of combination of the stealth action by polyethylene glycol cyanoacrylate-co-hexadecyl cyanoacrylate (PEG-PHDCA) modified niosomes and active targeting function of transferrin (Tf) by transferrin receptor-mediated endocytosis to promote drug delivery to solid tumor following intravenous administration with hydroxycamptothecin (HCPT) as model drug. HCPT-loaded PEG-niosomes (PEG-NS) were prepared by thin-film hydration and ultrasound method; the periodate-oxidated Tf was coupled to terminal amino group of PEG to produce the active targeting vesicles with average diameters of 116 nm. The uptake of Tf-PEG-NS into KB cells was concentration and time dependent, which could be inhibited by low temperature and free Tf, indicating that the endocytosis process was energy-driven and receptor specific. Compared with HCPT injection, non-stealth niosomes and PEG-NS, Tf-PEG-NS demonstrated the strongest cytotoxicity to three carcinomatous cell lines (KB, K562 and S180 cells), the greatest intracellular uptake especially in nuclei, the highest tumor concentration and largest area under the intratumoral hydroxycamptothecin concentration curve, as well as the most powerful anti-tumor activity with the inhibition rate of 71% against S180 tumor in mice. The results showed that the transferrin modified PEGylated niosomes could be one of the promising solutions to the delivery of anti-tumor drugs to tumor.

Novel anti-tumor strategy: PEG-hydroxycamptothecin conjugate loaded transferrin-PEG-nanoparticles.

The aim of the study was to prepare transferrin modified stealth nanoparticles (Tf-PEG-NP) encapsulating poly(ethylene) glycol-hydroxycamptothecin conjugate (PEG-HCPT) and exploit the possiblility of combination of the functions of passive and active targeting by Tf-PEG-NP, as well as sustained drug release in tumor by PEGylated drug for most efficient tumor targeting and anti-tumor effects enhancement. PEG was covalently linked to the 10-hydroxyl group of HCPT to produce PEG-HCPT conjugate. The conjugate was stable, highly water soluable with the cytotoxicity similar to the parent drug. By encapsulation of the drug conjugate in active targeting, long circulating nanoparticles, we further improved its therapeutic efficacy. The prepared Tf-PEG-NP with average diameters of 110nm showed more sustained in vitro release profile. The pharmacokinetic and biodistribution studies found that Tf-PEG-NP demostrated the longest retention time in blood (8.94-fold that of PEG-HCPT), the highest tumor accumulation (9.03-fold, 3.11-fold that of PEG-HCPT and HCPT-loaded counterpart, respectively), as well as the most powerful anti-tumor activity with the inhibition rate up to 93% against S180 tumor in mice (1.85-fold, 1.23-fold that of PEG-HCPT and HCPT-loaded counterpart, respectively). Such Tf-PEG-NP loaded with PEGylated drug conjugates could be one of the promising strategies to deliver anti-tumor drugs to tumor.

RGD-modified PEG–PAMAM–DOX conjugates: In vitro and in vivo studies for glioma

This work was based on our recent studies that a promising conjugate, RGD-modified PEGylated polyamidoamine (PAMAM) dendrimer with doxorubicin (DOX) conjugated by acid-sensitive cis-aconityl linkage (RGD-PPCD), could increase tumor targeting by binding with the integrin receptors overexpressed on tumor cells and control release of free DOX in weakly acidic lysosomes. To explore the application of RGD-PPCD to glioma therapy, the effects of the conjugate were further evaluated in glioma model. For comparative studies, DOX was also conjugated to PEG-PAMAM by acid-insensitive succinic linkage to produce the PPSD conjugates, which was further modified by RGD to form RGD-PPSD. In vitro cytotoxicity of the acid-sensitive conjugates against C6 cells was higher than that of the acid-insensitive ones, and further the modification of RGD enhanced the cytotoxicity of the DOX-polymer conjugates as a result of the increased cellular uptake of the RGD-modified conjugates by C6 cells. In vivo pharmacokinetics, biodistribution and antitumor activity were investigated in an orthotopic murine model of C6 glioma by i.v. administration of DOX-polymer conjugates. In comparison with DOX solution, all the conjugates showed significantly prolonged half-life and increased AUC and exhibited higher accumulation in brain tumor than normal brain tissue. Although RGD-PPCD was more than 2-fold lower tumor accumulation than RGD-PPSD, it exhibited the longest survival times among all treatment groups, and therefore, RGD-PPCD conjugate provide a desirable candidate for targeted therapy of glioma.

RGD-modified PEG-PAMAM-DOX conjugate: in vitro and in vivo targeting to both tumor neovascular endothelial cells and tumor cells.

Selective targeting of anticancer drugs to the tumor site is critical for effective cancer therapy. The cell adhesion molecule integrin α v β 3 is not readily detectable in quiescent vessels but highly expressed in angiogenic vessels and tumor cells. [ 1 ] This restricted expression profi le and good accessibility of integrin α v β 3 make it an ideal target for drug delivery purposes. [ 2 ] A number of synthetic cyclized arginine–glycine–aspartic acid sequences (RGDs) containing peptides, such as RGDyK, RGDfK, RGDfV, and RGDyV, have been identifi ed to have high affi nity with integrin α v β 3 . There has been growing interest in the synthesis and utilization of polymer–RGD conjugates for drug delivery, [ 3 ] gene delivery, [ 4 ] and imaging applications. [ 5 ]

PEGylated PAMAM dendrimers as a potential drug delivery carrier: in vitro and in vivo comparative evaluation of covalently conjugated drug and noncovalent drug inclusion complex

To understand more about the influence of the types of interaction between drug and PEGylated PAMAM dendrimers on the in vitro and in vivo behavior of drug, methotrexate (MTX) was coupled to PEGylated or non-PEGylated generation 4 PAMAM (G4) through complexing drug within the dendritic architecture and covalently conjugated onto the surface of the dendrimer, respectively. PAMAM was first modified with PEG5000 chains at three different degrees of substitution. The ability of PEGylated G4 complexing MTX was higher than that of non-PEGylated one. MTX-G4 and MTX-G4-PEG conjugates were synthesized via amide linkages. MTX was readily released from all complexes in isotonic solution, while the conjugates hardly released MTX in the same medium and keep stable in human plasma and the lysosomal medium. There were no obvious differences between complexes and free MTX in cytotoxicity against KB cell line, whereas the conjugates showed the relatively low activity. In vivo study in rodents found that the MTX-G4-PEG conjugate exhibited significantly prolonged blood residence time and the strongest antitumor effects, as compared with MTX-G4, the complexes and MTX. The results indicated that the covalent attachment of drug to PEGylated PAMAM could be more effective for targeted drug delivery.

Effects of polyoxyethylene (40) stearate on the activity of P-glycoprotein and cytochrome P450

The present study was aimed to investigate the effects of polyoxyethylene (40) stearate (PS), a non-ionic surfactant, on the activity of P-glycoprotein (P-gp) and six major cytochrome P450 (CYP) isoforms. An in vitro diffusion chamber system was utilized to estimate the effects of PS concentration on the transport characteristics of Rhodamine 123 (R123) and Rhodamine 110 (R110), a standard P-gp substrate and nonsubstrate, respectively, across the excised intestinal segments of rat. Caco-2 cells were cultured to investigate the mechanisms by estimating the effects of PS on intracellular ATP levels, P-gp ATPase activity and membrane fluidity. The obtained results showed that PS inhibited P-gp mediated efflux in a concentration-dependent manner mainly by modulating substrate-stimulated P-gp ATPase activity. On the other hand, human liver microsomes were utilized to examine the inhibitive potential of PS on six major CYP isoforms. Inhibitive potential on two of these CYP2C9 and CYP2C19 was found to be clinically significant. In conclusion, PS is potentially useful as a pharmaceutical ingredient to improve the oral bioavailability of coadministered P-gp substrates and substrates for certain CYP isoforms.

Application of Box-Behnken design in understanding the quality of genistein self-nanoemulsified drug delivery systems and optimizing its formulation

The purpose of the present study was to understand the effect of formulation variables of self- nanoemulsified drug delivery systems (SNEDDS) on the rapid dissolution of a model drug, genistein (GN). A three-factor, three-level Box-Behnken design was used to explore the main and interaction effect of several independent formulation variables including the amount of Maisine 35-1 and Labrafac Lipophile WL 1349 (1:1, w/w) (X1), Cremophor EL and Labrasol (3:1, w/w) (X2), and Transcutol P (X3). Droplet size (Y1), turbidity (Y2), and dissolution percentage of GN after 5 (Y3) and 30 (Y4) min were the dependent variables. A mathematical relationship, Y3 = − 89.3447 + 5.9524X1 + 1.0683X2 + 0.462X3 − 0.0825X12 − 0.0075X22 − 0.0009X32 + 0.0104X1X2  − 0.0113X1X3 + 0.0009X2X3 (r2 = 0.9604), was obtained to explain the effect of all factors and their co-linearities on the dissolution of GN at 5 min. Formulation optimization was then performed to maximize dissolution percentage of GN at 5 min (Y3). The optimized formulation was predicted to dissolution 93.34% of GN at 5 min, when X1, X2 and X3 values were 37.1, 101.7 and 77.3 mg, respectively. A new batch was prepared according to the optimized formulation, and the observed and predicted values of Y3 were in close agreement. In conclusion, the Box-Behnken experimental design allowed us to understand the effect of formulation variables on the rapid dissolution of GN from SNEDDS, and optimize the formulation to obtain a rapid drug dissolution at 5 min.

Active tumor-targeted delivery of PEG-protein via transferrin–transferrin-receptor system

Transferrin (Tf) holds promise as a drug carrier because of overexpress transferring receptors (TfRs) on the surface of tumor cells. The purpose of this work was to conjugate Tf to PEGylated protein (Tf–PEG-protein) to improve tumor-targeted delivery of PEG-protein. After a model protein, β-lactoglobulin B (LG), was modified by the heterobifunctional polyethyleneglycol (PEG), Tf was covalently linked to the distal end of the PEG chains on the PEG–LG (PL) conjugate. The purified Tf–PEG–LG (TPL) contained 1.4 of Tf ligand on one LG molecule. The specificity and affinity of TPL to TfR on two kinds of tumor cells (K562 and KB cells) were assessed. The results demonstrated that, TPL can bind specifically to the TfR on the tumor cell surface and the affinity of the conjugate to TfR was similar to that of native Tf. The pharmacokinetics and biodistribution studies in rodents found that TPL exhibited a significantly delayed blood clearance, the longest tumor resident time and the greatest tumor accumulation, as compared with LG and PL. Such design of the Tf conjugate would suggest a promising approach for active tumor targeting of therapeutic proteins or peptides to target cells.