Heparin-Regulated Prodrug-Type Macromolecular Theranostic Systems for Cancer Therapy

Abstract

Heparin is a kind of naturally occurring polymer with excellent biocompatibility and solubility. It is characterized by dense of negative charge, higher than any endogenous components. Heparin can bind with various cationic peptides and proteins, thereby providing a useful noncovalent linkage for building a drug delivery system. As a case in point, heparin/cell-penetrating peptides (CPP) interaction is strong, and remains stable in vivo. They can be used to modify different proteins, respectively, and subsequently, by simply mixing the modified proteins, a protein-protein conjugate can be form via the stable heparin/CPP linkage. This linkage could not be broken unless addition of protamine that bears higher cationic charge density than CPP, and CPP thus can be substituted and released. Of note, heparin is a potent antagonist of CPP, and their binding naturally inhibits CPP-mediated drug cell penetration. Based on this method, we developed a heparin-regulated macromolecular prodrug-type system, termed ATTEMPTS, for drug targeting delivery. In this review article, we mainly summary the application of ATTEMPTS in delivery of various macromolecular drugs for cancer therapy, and also introduce the heparin-regulated nanoprobes for tumor imaging.

Keywords: Heparin; drug targeting delivery; heparin-regulated macromolecular prodrug-type system.

Fig 1

The chemical structure of heparin unit.

Fig 2

Heparin-assisted “ATTEMPTS” for site-specific delivery of PA targeting to fibrin blots with reduced risk of hemorrhage.

Fig 3

(A) Inhibition of tPA activity by the heparin-antifibrin IgG and reversal by protamine. (B) In vitro clot lysis studies. Wells 1-4 contain 0.025, 0.05, 0.1 and 0.2 μg of tPA, respectively; well 5 contains 0.2 μg of free recombinant cationic tPA; well 6 contains 0.2 μg recombinant cationic tPA binding to the heparin complex (herein heparin beads were used); well 7 contains 0.2 μg recombinant cationic tPA triggering with 50 μg protamine; well 8 contains buffer only. Reproduced with permission from .

Fig 4

Extent of clot dissolution after administration of CM-tPA, CM-tPA/Hep-Ab, or CM-tPA/Hep-Ab plus protamine. The inferior vena cava of each rat was harvested for determination of clot weight. Reproduced with permission from . (Note: CM-tPA, cation-modified tPA)

Fig 5

(a) The construct of camouflaged tPA consisting of targeting peptide-HSA-protamine and tPA-low molecular-weight heparin (tPA-LMWH). (b) The albumin bound with tPA will provide steric hindrance to tPA-binding macromolecules in plasma. (c) Deposition of the complex on the surface of the activated platelets associated with the thrombus via peptide-glycoprotein (GP) IIb/IIIa binding; (d) Administration of heparin after accumulation of the complex at thrombus site; (e) Triggered release of tPA for local plasminogen activation and fibrinolysis. Reproduced with permission from .

Fig 6

In vivo clot lysis in rat jugular-vein thrombosis model. (a) Extent of clot lysis after administration of tPA and heparin combination (tPA-Hep), camouflaged tPA and camouflaged tPA plus heparin. (b) Fold increase in aPTT 1 h after the treatment compared to the value measured before the intervention. Data represent mean ± SD, n = 3, *p < 0.05,**p < 0.01. Reproduced with permission from

Fig 7

The modified ATTMEPTS with a triggerable cell-penetrating ability. The system consists of a targeting component (Ab-heparin) and drug component (drug-CPP), which spontaneously associate with each other via the interaction of heparin-CPP. Following administration, the prodrug feature and targeting function can reduce non-specific cell penetration and alleviate side effects on normal tissues. In the targeted tumor, drug component would be released by triggering with protamine, and its cell-penetrating ability recovered. The cleavable S-S linkage was designed in favor of retaining the protein drug inside the cytosol. Reproduced with permission from .

Fig 8

Cell uptake study results of rGel and TAT-Gel on LS174T cells. The cells were incubated with either TRITC-labeled (A) rGel or (B) TAT-Gel for 3 h at 37°C. After incubation, the cells were washed for three times with 10 mg/mL heparin/PBS solution for stringent wash, counterstained the nuclei with Hoechst 33342, and, after three more wash with PBS, the cell images were taken by a confocal microscope utilizing TRITC (red), Hoechst (blue) channels and merged. (rGel: recombinant gelonin, TAT-Gel: recombinant TAT-gelonin fusion protein). Reproduced with permission from .

Fig 9

T84.66-Hep/protamine-mediated modulation of cytotoxicity by TAT-Gel. (A) T84.66-Hep blocking of TAT-Gel cell transduction. When LS174T cells were incubated with TAT-Gel/T84.66-Hep complex, prepared by mixing TAT-Gel with increasing TAT-to-Hep molar ratios (from 5:1 to 1:3) of T84.66-Hep, a significantly reduced cytotoxicity was observed, compared with that of TAT-Gel. (B) Protamine-induced release of TAT-Gel. Addition of protamine to TAT-Gel/T84.66-Hep-treated cells, with increasing Hep-to-Pro molar ratios (from 10:1 to 1:2), cytotoxicity effects were significantly augmented. At above 5:1 molar ratio, the anti-cancer effect (IC50) was similar to that induced by TAT-Gel alone. (C) Time dependency of the protamine addition time on effects of protamine-triggered release. Cells were incubated with TAT-Gel/T84.66-Hep for 6 h, and, after wash, at intended time points (0, 2, 6, 24 and 48 h), protamine (Hep : Pro molar ratio of 1:2) was added to the wells, and the cells were incubated with protamine further up to total 72 h. ***P < 0.001 and n.s.: not significant by 1-way ANOVA (Tukey's multiple comparison test as the post hoc test). (TAT-Gel: recombinant TAT-gelonin fusion chimera, T84.66-Hep: T84.66-heparin chemical conjugate, Hep: heparin, Pro: protamine). Reproduced with permission from .

Fig 10

In vivo proof-of-concept efficacy study for assessment of the feasibility of PTD-modified ATTEMPTS using LS174T xenograft tumor bearing mice. (A) Tumor volumes (mm3) at day 40 (40 days after tumor implantation). At day 3, mice were divided into 5 groups (N = 10) and treated with either: 1) PBS, 2) TAT-Gel, 3) TAT-Gel/T84.66-Hep, 4) “TAT-Gel/T84.66-Hep+Pro” or 5) protamine (a.k.a. Pro) for three times (at day 3, 6 and 9) via tail vein injection. (B) Representative mice and tumor images at day 40. (C) Relative average body weight change (%) of mice during efficacy study. “TAT-Gel/T84.66-Hep+Pro” treatment exerted significantly enhanced therapeutic effects (*P < 0.05), compared with TAT-Gel/T84.66-Hep treatment, yet induced higher toxicity. *P < 0.05, **P < 0.01 and ***P < 0.001 by 1-way ANOVA (Tukey's multiple comparison test as the post hoc test). (TAT-Gel: recombinant TAT-gelonin fusion chimera, T84.66-Hep: T84.66-heparin chemical conjugate, TAT-Gel/T84.66-Hep+Pro”: treatment with TAT-Gel/T84.66-Hep complex with protamine). Reproduced with permission from .

Fig 11

Fluorescence microscopy of: (A) FITC-ASNase; (B) FITC-TAT-ASNase; (C) FITC-TAT-ASNase with heparin; and (D) FITC-TAT-ASNase with heparin and protamine. HeLa cells were incubated with the different treatment groups. Reproduced with permission from .

Fig 12

Flow cytometry analysis of FITC-TAT-ASNase conjugate. (A) ASNase and TAT-ASNase were FITC-labeled and incubated with MOLT-4 cells. (B) FITC-TAT-ASNase was incubated with heparin or heparin and protamine in MOLT-4 cells. Reproduced with permission from .

Fig 13

Schematic illustration of the synthesis of CPT-PR-LMWP conjugates. Reproduced with permission from .

Fig 14

Cellular localization of Rho-labeled LMWP-drug conjugates in A2780 human ovarian carcinoma cells. Free CPT, Rho-labeled CPT-PR, Rho-labeled CPT-PR-LMWP, Rho-labeled CPT-PR-LMWP and heparin mixture, and Rho-labeled CPT-PR-LMWP, heparin, and protamine mixture were overlaid onto cultured A2780 cells in the presence of 10% FBS. Cellular localization was monitored by confocal microscopy. (1) Free CPT; (2) Rho-labeled CPT-PR; (3) Rho-labeled CPT-PR-LMWP; (4) Rho-labeled CPT-PR-LMWP and heparin mixture; (5) Rho-labeled CPT-PR-LMWP, heparin, and protamine mixture. (A) 430 nm (blue) detection; (B) 560 nm (red) detection; (C) overlaid (A + B). Reproduced with permission from .

Fig 15

The schematic illustration of the legumain-activatable nanoprobe for tumor imaging. Reproduced with permission from .

Fig 16

The cellular activation process of the nanoprobe. Reproduced with permission from .

Fig 17

The in vivo imaging of the MMP-activatable nanoprobe in the subcutaneous HT1080 tumor model. Reproduced with permission from .

Fig 18

(A) Tumor-associated activation of the nanoprobe. (B) Blood vessel staining (green) and the activated nanoprobe (red) in HT1080 tumor. (C) Western blotting analysis of MMP-2 in HT1080, MCF-7, and SW620 tumor. Reproduced with permission from .

Fig 19

(A) The orthotopic glioma imaging with the T7-functionalized nanoprobe at various time points. (B) The radiant efficiency (fluorescence intensity). Reproduced with permission from .

Fig 20

(A) The tumor volume of PBS, SAHA-L, LHT7, the sequential co-administration of SAHA-L and LHT7, or LHT7/SAHA nanolipoplex groups treated. (B) Comparative tumor mass after 14 days. Reproduced with permission from