Development of Liposomal Gemcitabine with High Drug Loading Capacity

Abstract

Liposomes are widely used for systemic delivery of chemotherapeutic agents to reduce their nonspecific side effects. Gemcitabine (Gem) makes a great candidate for liposomal encapsulation due to the short half-life and nonspecific side effects; however, it has been difficult to achieve liposomal Gem with high drug loading capacity. Remote loading, which uses a transmembrane pH gradient to induce an influx of drug and locks the drug in the core as a sulfate complex, does not serve Gem as efficiently as doxorubicin (Dox) due to the low p K_a value of Gem. Existing studies have attempted to improve Gem loading capacity in liposomes by employing lipophilic Gem derivatives or creating a high-concentration gradient for active loading into the hydrophilic cores (small volume loading). In this study, we combine the remote loading approach and small volume loading or hypertonic loading, a new approach to induce the influx of Gem into the preformed liposomes by high osmotic pressure, to achieve a Gem loading capacity of 9.4-10.3 wt % in contrast to 0.14-3.8 wt % of the conventional methods. Liposomal Gem showed a good stability during storage, sustained-release over 120 h in vitro, enhanced cellular uptake, and improved cytotoxicity as compared to free Gem. Liposomal Gem showed a synergistic effect with liposomal Dox on Huh7 hepatocellular carcinoma cells. A mixture of liposomal Gem and liposomal Dox delivered both drugs to the tumor more efficiently than a free drug mixture and showed a relatively good anti-tumor effect in a xenograft model of hepatocellular carcinoma. This study shows that bioactive liposomal Gem with high drug loading capacity can be produced by remote loading combined with additional approaches to increase drug influx into the liposomes.

Keywords: drug loading capacity; gemcitabine; hypertonic loading; liposomes; remote loading; small volume loading.

Fig. 1.

Schematic description of the liposome preparation. (a) Overview of passive loading, remote loading, small volume loading, and hypertonic loading. (b-d) Envisioned mechanism of each method. d-N represents weak base drug such as Gem or Dox.

Fig. 2.

TEM images of L_RSG, L_RHG, blank L_RH, and blank L_RS. Scale bar: 200 nm.

Fig. 3.

In vitro release kinetics of (a) L_RSG, L_RHG and (b) L_RD at pH 5.5 and 7.4. n = 3 independent and identical batches. Mean ± standard deviation (s.d.).

Fig. 4.

Cytotoxicity of (a) free Gem, L_RSG, and L_RHG and (b) free Dox and L_RD after 72 h incubation. n = 3 identical and independent tests. Mean ± s.d.

Fig. 5.

Cytotoxicity of (a) free Gem, L_RSG, and L_RHG and (b) free Dox and L_RD after short-term incubation. n = 5 tests of a representative batch. Mean ± s.d. Drug uptake by Huh7 after 3, 6, 12 and 24 h incubation with (c) free Gem, L_RSG, and L_RHG and (d) free Dox and L_RD. n = 3 (Gem) and 4 (Dox) independent and identical tests of a representative batch. Mean ± s.d. *: p < 0.05, **: p < 0.01 by, ****: p < 0.0001 by two-way ANOVA test followed by Sidak’s multiple comparisons test.

Fig. 6.

Confocal microscopic images of Huh7 cells incubated with (a) free Dox or (b) 25-NBD cholesterol labeled *L_RD for 10 min, 3 h or 10 h. + CQ: cells incubated with chloroquine (inhibitor of endosomal acidification) for 12 h prior to the addition of liposomal Dox. Scale bars: 50 μm.

Fig. 7.

Cytotoxicity of (a) free Gem/Dox combinations on Huh7 cells given in different sequences and in different molar ratios (n = 3 tests. Mean ± s.d.) and (b) free or liposomal Gem/Dox combinations on Huh7 cells given simultaneously in different molar ratios (n = 3 tests. mean ± s.d.).

Fig. 8.

Drug concentrations in (a) plasma and (b) Huh7 tumors of animals treated with Gem + Dox free drug mixture or L_RSG + L_RD liposomal mixture at 8 h post-IV injection. n = 4 expect for the plasma of liposomal mixture-treated group: n = 3.

Fig. 9.

(a) Dosing schedule. (b) Changes of individual Huh7 tumor volume. Blue: PBS (n = 6); Red: Gem + Dox (n = 8); Green: L_RSG + L_RD (n = 7). (c) Specific growth rate of Huh7 tumor: ΔlogV/Δt (V: tumor volumes; t: time in days). **: p < 0.01, ***: p <0.001 by Tukey’s multiple comparisons test. (d) Survival curve of the animals receiving PBS, Gem + Dox, or L_RSG + L_RD. ***: p <0.001, ****: p <0.0001 by Log-rank (Mantel-Cox) test.

Fig. 10.

(a) Representative photographs of TUNEL-stained Huh7 tumor sections and quantitative analysis of TUNEL-stained sections. Top: TUNEL-stained apoptotic cells; Bottom: Composite images of TUNEL-stained apoptotic cells (green) and PI-stained nuclei (red); % apoptotic cells = number of apoptotic cells/total number of nuclei measured by a Nikon A1R confocal microscope (3–4 random fields per each tumor section). Scale bars: 100 μm. **: p < 0.01 vs. PBS by Dunn’s multiple comparisons test following Kruskal-Wallis test. For all images, see Supporting Fig. 8; (b) Representative H&E stained Huh7 tumor sections. Scale bars: 50 μm. For other organs, see Supporting Fig. 9.