Construction and in vivo / in vitro evaluation of a nanoporous ion-responsive targeted drug delivery system for recombinant human interferon α-2b delivery

Abstract

Background: Like most protein macromolecular drugs, the half-life of rhIFNɑ-2b is short, with a low drug utilization rate, and the preparation and release conditions significantly affect its stability.

Methods: A nanoporous ion-responsive targeted drug delivery system (PIRTDDS) was designed to improve drug availability of rhIFNα-2b and target it to the lung passively with sustained release. Chitosan rhIFNα-2b carboxymethyl nanoporous microspheres (CS-rhIFNα-2b-CCPM) were prepared by the column method. Here, an electrostatic self-assembly technique was undertaken to improve and sustain rhIFNα-2b release rate.

Results: The size distribution of the microspheres was 5~15 μm, and the microspheres contained nanopores 300~400 nm in diameter. The in vitro release results showed that rhIFNα-2b and CCPM were mainly bound by ionic bonds. After self-assembling, the release mechanism was transformed into being membrane diffusion. The accumulative release amount for 24 hrs was 83.89%. Results from circular dichrogram and SDS-PAGE electrophoresis showed that there was no significant change in the secondary structure and purity of rhIFNα-2b. Results from inhibition rate experiments for A549 cell proliferation showed that the antitumor activity of CS-rhIFNα-2b-CCPM for 24 hrs retained 91.98% of the stock solution, which proved that the drug-loaded nanoporous microspheres maintained good drug activity. In vivo pharmacokinetic experimental results showed that the drugs in CS-rhIFNα-2b-CCPM can still be detected in vivo after 24 hrs, equivalent to the stock solution at 6 hrs, which indicated that CS-rhIFNα-2b-CCPM had a certain sustained-release effect in vivo. The results of in vivo tissue distribution showed that CS-rhIFNα-2b-CCPM was mainly concentrated in the lungs of mice (1.85 times the stock solution). The pharmacodynamics results showed that CS-rhIFNα-2b-CCPM had an obvious antitumor effect, and the tumor inhibition efficiency was 29.2%.

Conclusion: The results suggested a novel sustained-release formulation with higher drug availability and better lung targeting from CS-rhIFNα-2b-CCPM compared to the reference (the stock solution of rhIFNα-2b), and, thus, should be further studied.

Keywords: ion exchange technique; ion-responsive; lung targeting; nanoporous; recombinant human interferon α-2b; sustained release.

Figure 1

The mechanism of CCPM loading and releasing drugs by ion exchange. Abbreviation: CCPM, carboxymethyl chitosan nanoporous microspheres.

Figure 2

The diagram of the dynamic loading drug process. Abbreviation: CCPM, carboxymethyl chitosan nanoporous microspheres.

Figure 3

Schematic diagram of the electrostatic self-assembly technique. Abbreviations: rhIFNα-2b-CCPM, rhIFNα-2b carboxymethyl chitosan nanoporous microspheres; CS-rhIFNα-2b-CCPM, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres.

Figure 4

The effect of time on permeability and drug utilization rate of the drug-loading process (n=3).

Figure 5

Characterization studies. (A) SEM images (ⅰ: CCPM; ⅱ: surface of CCPM; ⅲ: rhIFNα-2b-CCPM; ⅳ: CS-rhIFNα-2b-CCPM); (B) Particle size distribution of CS-rhIFNα-2b-CCPM; (C) Accumulative release from the optimal formulation; (D) Electrophoretogram of different rhIFNα-2b samples dyed by Coomassie brilliant blue (a: rhIFNα-2b solution; b: rhIFNα-2b extracted from nanoporous microspheres; c: rhIFNα-2b release solution for 12 hrs; d: rhIFNα-2b extracted from nanoporous microspheres after in vitro release for 24 hrs; e: rhIFNα-2b in effluent and washing liquid); (E) Circular dichroism spectra of rhIFNα-2b; (F) Inhibition rate of cell proliferation (n=3); and (G) Micrograph of the inhibition effect of the nanoporous microsphere releasing solution with different concentrations on A549 cells. Abbreviations: CCPM, nanoporous microspheres; rhIFNα-2b-CCPM, rhIFNα-2b carboxymethyl chitosan nanoporous microspheres; CS-rhIFNα-2b-CCPM, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres; IR, inhibition rate.

Figure 6

Drug concentration–time data following i.v. administration of the rhIFNα-2b solution and nanoporous microspheres in mice (n=3). Abbreviation: CS-rhIFNα-2b-CMCS-MS, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres.

Figure 7

Tissue distribution diagram after tail vein injection (ng·g⁻¹) (n=3). (A) histogram of drug concentration in each tissue after 0.5, 1, 4, and 6 hrs after rhIFNα-2b stock solution injection into the tail vein of mice. (B) Histogram of drug concentration in various tissues after the injection of rhIFNα-2b nanoporous microspheres into the tail vein of mice after 1, 4, 6, and 24 hrs.

Figure 8

The evaluation of the antitumor effect (n=5). (A) The appearance of the tumors. (B) Histopathology slice of tumor (i: the control group; ii: the stock solution group; and iii: the microsphere group). Abbreviation: CS-rhIFNα-2b-CCPM, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres.