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

Figures & data

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. 

Table 1 Drug loading and encapsulation rate of the optimized prescription of CS-rhIFNα-2b-CCPM (n=3)

Batch	1	2	3	Mean	SD	RSD%
tDL(μg·mg^−1)	15.01	9.36	11.54	11.97	2.85	23.80
rDL(μg·mg^−1)	13.55	8.42	10.33	10.77	2.59	24.08
rE (%)	90.27	89.96	89.51	89.91	0.38	0.43

Abbreviation: CS-rhIFNα-2b-CCPM, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres.

Table 2 Results of the drug release behavior fitted by different kinetic models. (mean±SD, n=6)

Kinetic model	rhIFNα-2b-CCPM		CS-rhIFNα-2b-CCPM
	k±SD	r^2	k±SD	r^2
Zero-order model	3.0206±0.29	0.7286	2.936±0.45	0.9462
First-order model (film diffusion)	0.0841±0.017 hr^−1	0.9406	0.0603^1±0.0019 hr^−	0.9986
Higuchi model (matrix diffusion)	17.168±1.62 hr^−1/2	0.9253	17.023±2.38 hr^−1/2	0.9852
Hixson and Crowell cube-root model	0.0297±0.0098 hr^−3	0.8824	0.0717±0.013 hr^−3	0.9909
Viswanathan	0.264±0.055	0.9918	0.198±0.073	0.9759

Abbreviations: rhIFNα-2b-CCPM, rhIFNα-2b carboxymethyl chitosan nano-porous microspheres; CS-rhIFNα-2b-CCPM, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres.

Table 3 Results of the recovery test (n=3)

Tissues	C (pg/mL)	Recovery (%)			Mean±SD（%）	RSD (%)
		1	2	3
Plasma	15	98.41	82.75	89.71	90.29±6.41	8.69
	60	87.68	93.32	96.54	92.51±3.66	4.85
	240	97.51	89.31	93.2	93.34±3.35	4.39
Liver	15	96.67	85.17	77.65	86.50±7.82	11.07
	60	87.39	93.71	87.42	89.51±2.97	4.07
	240	95.65	96.28	89.16	93.70±3.22	4.21
Lung	15	89.68	97.32	85.54	90.85±4.88	6.58
	60	94.41	85.75	94.71	91.62±4.15	5.55
	240	87.51	93.31	98.2	93.01±4.37	5.75
Kidney	15	86.09	85.02	99.01	90.04±6.36	8.65
	60	93.8	79.62	87.11	86.84±5.79	8.17
	240	95.39	88.44	90.2	91.34±2.95	3.96
Heart	15	95.23	81.15	86.34	87.57±5.81	8.13
	60	77.32	94.12	91.33	87.59±7.35	10.28
	240	90.87	85.77	83.61	86.75±3.04	4.30
Spleen	15	85.68	89.15	93.7	89.51±3.28	4.49
	60	90.32	84.32	98.54	91.06±5.83	7.84
	240	86.54	90.22	97.43	91.40±4.52	6.06
Brain	15	98.28	95.54	84.11	92.64±6.14	8.11
	60	91.99	94.16	85.01	90.39±3.90	5.29
	240	85.08	89.99	94.34	89.80±3.78	5.16

Table 4 Pharmacokinetic parameters in mice plasma (mean±SD, n=3)

Parameter	rhIFNα-2b	CS-rhIFNα-2b-CCPM
Tmax (h)	0.08±0.02	1.5±0.03
Cmax (pg·mL^−1)	3797±785	846±222
MRT0-∞ (h)	0.8±0.2	12.0±2.2
t1/2 (h)	0.8±0.2	9.0±1.5
AUC0-∞ (pg·L^−1·h)	2488±1132	2159±747
Relative bioavailability (F%)	/	86.8±6.6

Abbreviations: rhIFNα-2b-CCPM, rhIFNα-2b carboxymethyl chitosan nanoporous microspheres; CS-rhIFNα-2b-CCPM, chitosan rhIFNα-2b carboxymethyl chitosan nanoporous microspheres.

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.

Table 5 Target index in mice tissues (mean ± SD, n=3)

Tissues	Ci (pg/mL)		Ce
Solution	Nanoporous microspheres
Lung	283.36±32.89	522.98±43.93	1.85±0.65
Kidney	582.71±67.56	382.05±25.37	0.66±0.11
Liver	71.59±9.97	80.63±17.66	1.13±0.38
Heart	142.88±23.35	142.79±13.28	1.00±0.14
Spleen	77.29±8.59	68.14±9.25	0.88±0.21
Brain	20.72±5.92	15.25±4.27	0.73±0.21

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.