Chitosan-dipotassium orthophosphate lyophilizate: a novel in situ thermogel carrier system of allogeneic platelet lysate growth factors

Figures & data

Table 1. Formulations tested at different CS and DHO concentrations.

Formula	CS Conc. (%w/v)	DHO Conc. (%w/v)
F1	1.8	1.5
F2	1.8	2.0
F3	1.8	2.5
F4	1.4	1.5
F5	1.4	2.0
F6	1.4	2.5

Table 2. Freeze-drying protocols investigated for F3 at a fixed Man: Suc of 6:2%w/v.

	Freezing		Annealing		Primary Drying		Secondary Drying
FD protocols	Temperature (°C)	Duration (hrs)	Temperature (°C)	Duration (hrs)	Temperature (°C)	Duration (hrs)	Temperature (°C)	Duration (hrs)
Unannealed	−20	24	–	–	−54	12	–	–
Conservative	−45	18	−20	6	−54	12	–	–
Extensive	−45	18	−20	6	−54	12	ambient	2 hrs

Table 3. CQA outcomes for optimization of cryoprotectants levels and FD procedure.

Parameter	Critical Quality Attribute (CQA)
	Cake appearance	Residual moisture (%)	Reconstitution Product	Reconstitution time (min)
Man: Suc (% w/v)	All samples prepared using unannealed FD protocol
4:1	Granular with large aggregates	11.34 ± 3.63	Significant precipitation	N/A
4:2	Granular with large aggregates	9.81 ± 2.85	Significant precipitation	N/A
6:2	Granular	10.28 ± 2.40	Some precipitation	N/A
FD protocol	All samples prepared with fixed Man: Suc at 6/2 %w/v
Unannealed	Granular with large aggregates	10.28 ± 2.40	Some precipitation	N/A
Conservative	Fine powder	4.47 ± 1.13^a	Completely soluble	3.28 ± 0.49
Extensive	Fine powder	4.09 ± 0.91^b	Completely soluble	2.52 ± 0.81

^{a, b} Residual moisture content was lower than that of unannealed FD samples, ρ < .05.

Figure 1. Lyophilizate optimization and characterization. (A) Particulate sample subjected to suboptimal FD cycle, (B) Cake collapse with prominent ridge formation and (C) Optimized FD formulation with crystalline Man scaffold, (D) DSC thermogram of F3, (E) X-ray diffractograms of (i) Man and (ii) F3, and (F) FT-IR spectra for (i) HPL, (ii) CS and (iii) F3.

Table 4. pH and gelation time of medicated CS/DHO lyophilizates after reconstitution.

Formula	CS (%w/v)	DHO (%w/v)	pH	Gelation time
F1	1.8	1.5	7.32 ± 0.06	6.19 ± 2.74
F2	1.8	2.0	7.44 ± 0.13	5.14 ± 1.23
F3	1.8	2.5	7.58 ± 0.05	2.84 ± 0.66
F4	1.4	1.5	6.96 ± 0.08	15.55 ± 4.73
F5	1.4	2.0	7.15 ± 0.04	10.50 ± 1.26
F6	1.4	2.5	7.11 ± 0.16	9.26 ± 3.51

Figure 2. Representative procedure of the release experiment. (A) reconstitution of the lyophilizate and (B) The sample at the beginning (i), during (ii) and at the end (iii) of the release experiment.

Figure 3. Total protein release profile of F1 and F2 at pH 6.5 and 7.4.

Figure 4. Release profile for Total protein. (A) F1 vs F4, (B) F2 vs. F5, (C) F3 vs. F6, and (D) PDGF-BB F1 vs F4, (E) F2 vs. F5, (F) F3 vs. F6 at a fixed DHO concentration of 1.5, 2 and 2.5%w/v, respectively.

Table 5. Kinetic modeling of total protein and PDGF-BB release data.

Kinetic Model	Total Protein r^2	PDGF-BB r^2
Zero Order	0.9793	0.8697
First Order	0.96	0.9565
Higuchi	0.9838	0.9860
Korsmeyer Peppas	0.9976 (n = 0.4982)	0.9694 (n = 0.3865)
Weibull	0.9775	0.7835
Hickson Crowell	0.8253	0.7090

r² is the correlation coefficient and n is the release exponent.

Figure 5. Cytotoxicity testing of F3. Photomicrograph showing the viability of HSF cells cultured in direct contact with a CS/DHO gel prepared with 1.8% CS and 2% DHO at a HPL concentration of: (A) 0 μg/mL and (B) 100 μg/mL.

Data availability statement

The data that support the findings of this study are available from the corresponding author, Toaa A. Abdelrahman, upon reasonable request.