Combinatorial and sequential delivery of gemcitabine and oseltamivir phosphate from implantable poly(d,l-lactic-co-glycolic acid) cylinders disables human pancreatic cancer cell survival

Figures & data

Figure 1 Therapeutic design of OP- and GEM-loaded double-layered PLGA cylindrical implants.

Notes: Production of a double-layered PLGA. (A) Insoluble drug particles were suspended in the PLGA, Span 80, and acetone solution and ejected dropwise onto a Teflon sheet. (B) Acetone evaporates from the suspension, resulting in a thin, malleable film. (C) The film is scraped off the Teflon sheet using a razor blade and (D) wrapped around a glycerol-lubricated 16 G needle to form a cylinder. (E) The cylinder is then removed from the needle. A double-layered PLGA cylinder is formed by rolling a second film around the first cylinder. Adapted from Hrynyk M, Ellis JP, Haxho F, et al. Therapeutic designed poly (lactic-co-glycolic acid) cylindrical oseltamivir phosphate-loaded implants impede tumor neovascularization, growth and metastasis in mouse model of human pancreatic carcinoma. Drug Des Devel Ther. 2015;9:4573–4586.⁹
Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate; PLGA, poly(d,l-lactic-co-glycolic acid).

Figure 2 Double-layered PLGA cylinder with 16 mg OP inner layer and 3 mg GEM outer layer (A). Cross-section SEM micrographs of a control cylinder without drug (B) and cylinders containing 16 mg OP (C) and 3 mg GEM (D).

Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate; SEM, scanning electron microscopy.

Figure 3 (A) OP and GEM were detected using a 1260 Infinity HPLC and a Poroshell 120 SB-C18 threaded column with mobile phase 60% methanol and 40% 0.04 M ammonium acetate buffer (pH 5.2). Retention times of GEM and OP were 1.1 and 3.0 min, respectively. (B) Calibration curve for OP and GEM using HPLC.

Abbreviations: GEM, gemcitabine; HPLC, high performance liquid chromatograph; OP, oseltamivir phosphate.

Figure 4 Area under curve of peak representing OP in 0.1 M sodium phosphate buffer (pH 7.4) at 37°C (A). HPLC chromatograms of OP in sodium phosphate buffer at 37°C on day 0 (B) and day 30 (C). HPLC chromatogram of OC (D). HPLC chromatogram of GEM (left peak) and OP (right peak) still encapsulated in an OPⁱⁿ/GEM^out cylinder following 15 days in PANC1 cell culture (E).

Abbreviations: GEM, gemcitabine; OC, oseltamivir carboxylate; OP, oseltamivir phosphate.

Table 1 Formulations of PLGA cylinders containing OP and GEM and drug release from cylinders after 72 h in 0.1 M phosphate buffer (pH 7.4) at 37°C

Number of layers	PLGA (mg)	Layer arrangement, inner/outer	OP (mg)	GEM (mg)	Film storage	Release after 72 h (%)
1	80	–	16	3	24 h, 4°C	OP: 86 GEM: 61
	160	–	16	3	24 h, 4°C	OP: 86 GEM: 72
	320	–	16	3	24 h, 4°C	OP: 58 GEM: 31
2	160 (80 mg/layer)	OP/GEM	8	1.5	24 h, 4°C	OP: 3 GEM: 36
		GEM/OP	8	1.5	24 h, 4°C	OP: 12 GEM: 1
2	160 (80 mg/layer)	OP/GEM	16	3	24 h, 4°C	OP: 87 GEM: 18
		GEM/OP	16	3	24 h, 4°C	OP: 73 GEM: 3
2	160 (80 mg/layer)	OP/GEM	16	3	72 h, 20°C	OP: 21 GEM: 43
		GEM/OP	16	3	72 h, 20°C	OP: 53 GEM: 2

Notes: Double-layered PLGA cylinders in which the OP was contained within the inner cylinder and the GEM was contained within the outer cylinder are designated as OP/GEM, and cylinders in which the GEM was contained within the inner cylinder and the OP was contained within the outer cylinder are designated as GEM/OP. ‘–’ indicates not applicable as there was only one inner/outer layer arrangement.

Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate; PLGA, poly(d,l-lactic-co-glycolic acid).

Figure 5 Simultaneous release of OP and GEM from inner/outer for OPⁱⁿ/GEM^out (A) and GEMⁱⁿ/OP^out (B) double-layered PLGA cylinders.

Notes: Films were held at 20°C for 72 h prior to cylinder rolling. Data points represent the mean ± standard deviation, n=3 separate experiments. OP and GEM released are reported as percentage of the total amount of drug released after 30 days.
Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate.

Figure 6 Release profile of OP (•) and GEM (■) from inner/outer OPⁱⁿ/GEM^out (A) and GEMⁱⁿ/OP^out (B) double-layered PLGA cylinders in comparison to the projected theoretical release of OP (- - -) and GEM (—), which assumes a constant release rate over 30 days.

Notes: The projected theoretical release rate was calculated from injected treatment doses of the two drugs shown effective in either the stagnation or reduction of tumor volume in a heterotopic xenograft model of human pancreatic cancer in mice (unpublished data, Szewczuk’s lab, August 2016).
Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate.

Figure 7 (A) PANC1 untreated cells over 15 days, compared to PANC1 exposed to drug-free blank, and inner/outer OPⁱⁿ/GEM^out, or GEMⁱⁿ/OP^out cylinders. Scale bar: 508 µm. (B) Viable cell numbers are expressed as a percentage of the untreated cells. The data are a representation of one out of two independent experiments showing similar results.

Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate.

Figure 8 PANC1-GEMR untreated cells over 15 days compared to PANC1-GEMR cells exposed to drug-free blank and inner/outer OPⁱⁿ/GEM^out or GEMⁱⁿ/OP^out cylinders (A). Scale bar: 508 µm. Viable cell numbers are expressed as percentage of the untreated cells (B). Absence of a bar indicates that no viable cells were present. The data are the representation of one out of two independent experiments showing similar results.

Abbreviations: GEM, gemcitabine; OP, oseltamivir phosphate.

HrynykMEllisJPHaxhoFTherapeutic designed poly (lactic-co-glycolic acid) cylindrical oseltamivir phosphate-loaded implants impede tumor neovascularization, growth and metastasis in mouse model of human pancreatic carcinomaDrug Des Devel Ther20159 4573 4586

 PubMed Web of Science ®Google Scholar