We designed and synthesized new dextran-peptide-methotrexate conjugates for tumor-targeted delivery of chemotherapeutics via the mediation of matrix metalloproteinase II (MMP-2) and matrix metalloproteinase IX (MMP-9), both being widely known tumor-associated enzymes. A robust and flexible synthesis procedure and process monitoring chromatography assays were developed. The linker chemistry and the backbone charge were optimized to allow high sensitivity of the conjugates toward the targeted enzymes. The optimal conjugate carries Pro-Val-Gly-Leu-Ile-Gly as the peptide linker, and the charge on the dextran backbone is fully neutralized. In the presence of the targeted enzymes, the peptide was cleaved and peptidyl methotrexate was released, with a kcat/Km value of 1.21 x 10(5) M(-1) s(-1) for MMP-2 and 3.60 x 10(3) M(-1) s(-1) for MMP-9, respectively. Satisfactory stability of the new conjugates was demonstrated in serum containing conditions, suggesting the conjugates can remain intact in systemic circulation. These findings supported the tumor targeting capability of the new conjugates and warranted further investigation with in vivo study.

No abstract

We have designed a new dextran-peptide-methotrexate conjugate to achieve tumor-targeted delivery of chemotherapeutics. The dextran carrier was selected to allow passive targeting and enhanced permeation and retention (EPR). The peptide linker has also been optimized to allow drug release in the presence of matrix-metalloproteinase-2 (MMP-2) and matrix-metalloproteinase-9 (MMP-9), 2 important tumor-associated enzymes. The new conjugate was assessed for its in vivo antitumor efficacy and systemic side effects. It was compared with free methotrexate (MTX) and a similar conjugate, differing by an MMP-insensitive linker, at equivalent intraperitoneal dosages. The MMP-sensitive conjugate demonstrated tolerable in vivo side effects and effective inhibition of in vivo tumor growth by 83% in each of the 2 separate tumor models that overexpress MMP (HT-1080 and U-87). The antiproliferative effect of the drug contributed to the inhibition of tumor growth. In contrast, free MTX resulted in no significant tumor reduction in the same models. Neither free MTX nor the conjugate caused any tumor inhibition in the mice bearing RT-112, a slower growing model that does not overexpress MMP. MMP-insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow. ' 2005 Wiley-Liss, Inc.Key words: methotrexate; dextran; matrix metalloproteinase; gelatinase; tumor; enhanced permeation and retention (EPR); tumor-associated enzymes; polymer-drug conjugate; cancer targeting Polymer-drug conjugates have shown promise as drug-delivery vehicles for targeting low molecular weight drugs to tumor tissues. 1 By tailoring the polymeric carriers, the pharmacokinetics and tumor target ratio of the attached drug molecules can be favorably altered. The key to the success of this application is that covalent attachment to the polymer enables passive targeting of the drug molecules. The high molecular weight of the polymer increases the size of the conjugate and slows the drug clearance by the kidneys. The drug concentration in the plasma is thereby maintained above the therapeutic level for a prolonged period of time. Additionally, many solid tumors display unique pathophysiology features, including highly permeable vasculature and impaired lymphatic drainage, that are absent in normal tissues. High molecular weight polymer-drug conjugates extravasate into the tumor tissues but not the normal tissues with less permeable vessels. Once inside the tumor tissues, the polymer-drug conjugates do not readily return to the general circulation because of the poor lymphatics. This phenomenon, termed enhanced permeation and retention (EPR), was first discovered by Maeda. 2 To take advantage of passive targeting and EPR, we have considered the necessary criteria in our design of a new polymer-peptide-drug conjugate. The polymer backbone needs to be biocompatible and biodegradable. It should be hydrophilic, not highly charged, and should have a size above the renal threshold limit to increase its circulation time. C...

Increased uptake rate of folate-decorated NPs into RPE cells is observed with increasing degree of folate modification. These NPs utilize both clathrin- and caveolae-mediated receptor-mediated endocytosis pathways to enter RPE cells upon size variation. The 50 nm NPs are internalized the fastest, with clathrin-mediated endocytosis as the preferred route. Uptake of 250 nm particles is the slowest and is dominated by caveolae-mediated endocytosis.