New method for delivering a hydrophobic drug for photodynamic therapy using pure nanocrystal form of the drug

Abstract

A carrier-free method for delivery of a hydrophobic drug in its pure form, using nanocrystals (nanosized crystals), is proposed. To demonstrate this technique, nanocrystals of a hydrophobic photosensitizing anticancer drug, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH), have been synthesized using the reprecipitation method. The resulting drug nanocrystals were monodispersed and stable in aqueous dispersion, without the necessity of an additional stabilizer (surfactant). As shown by confocal microscopy, these pure drug nanocrystals were taken up by the cancer cells with high avidity. Though the fluorescence and photodynamic activity of the drug were substantially quenched in the form of nanocrystals in aqueous suspension, both these characteristics were recovered under in vitro and in vivo conditions. This recovery of drug activity and fluorescence is possibly due to the interaction of nanocrystals with serum albumin, resulting in conversion of the drug nanocrystals into the molecular form. This was confirmed by demonstrating similar recovery in presence of fetal bovine serum (FBS) or bovine serum albumin (BSA). Under similar treatment conditions, the HPPH in nanocrystal form or in 1% Tween-80/water formulation showed comparable in vitro and in vivo efficacy.

Figure 1

Structure of the HPPH

Figure 2

(A) TEM picture of HPPH nanocrystals (B) Electron diffraction from HPPH nanocrystals

Figure 3

Absorption and emission spectra of HPPH nanocrystals (4 μM) in comparison with Tween-80 micellar formulation at the same molar concentration in water.

Figure 4

(A) Fluorescence signal from HPPH nanocrystals (4 μM) in pure cell culture medium (MEMα) and MEMα with added FBS or BSA after 6 hrs of incubation at room temperature; (B) Time-dependent fluorescence signal recovery from HPPH nanocrystals (4 μM) in pure MEMα and MEMα with added FBS (10%v/v) or BSA (0.8% w/v).

Figure 5

Singlet oxygen phosphorescence decay monitored at 1270nm (1) HPPH nanocrystals in MEMα cell culture media and (2) HPPH nanocrystals incubated with MEMα and 0.8% BSA for 24 Hrs. The inset figure shows the corresponding fluorescence recovery. Concentration of HPPH was 45μM.

Figure 6

Confocal fluorescence images of RIF-1 cells imaged after 4 hrs, 24hrs and 48 hrs of treatment with HPPH nanocrystals (A) and tween-80 micellar formulation (B). Drug concentration was maintained at 0.5 μM. Cells were incubated in medium containing 10% of FBS.

Figure 7

Confocal fluorescence images of RIF-1 cells incubated with (A) Tween 80 micellar formulation and (B)HPPH nanocrystals, imaged after 2 hrs of incubation in serum free media. Excitation wavelength used was 405nm.

Figure 8

Comparative in vitro photosensitizing efficacy of HPPH formulated in1% Tween 80/5% dextrose and HPPH nanocrystals/water in RIF-1 cells at equimolar concentrations (0.5μM). Control: Cells were exposed to light without photosensitizer.

Figure 9

Comparison of in vivo photosensitizing efficacy of HPPH nanocrystals and Tween 80 micellar formulation in C3H mice (10 mice/group) bearing RIF-1 tumors. The tumors were exposed to a laser light (665 nm, 135 J/cm²) at 24 h after injection. Drug dose: 0.4 μ mole/kg. Control: The mice were injected with HPPH nanocrystals, without any light exposure.