Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles

Abstract

The paper reports the results of nanotherapy of ovarian, breast, and pancreatic cancerous tumors by paclitaxel-loaded nanoemulsions that convert into microbubbles locally in tumor tissue under the action of tumor-directed therapeutic ultrasound. Tumor accumulation of nanoemulsions was confirmed by ultrasound imaging. Dramatic regression of ovarian, breast, and orthotopic pancreatic tumors was observed in tumor therapy through systemic injections of drug-loaded nanoemulsions combined with therapeutic ultrasound, signifying efficient ultrasound-triggered drug release from tumor-accumulated nanodroplets. The mechanism of drug release in the process of droplet-to-bubble conversion is discussed. No therapeutic effect from the nanodroplet/ultrasound combination was observed without the drug, indicating that therapeutic effect was caused by the ultrasound-enhanced chemotherapeutic action of the tumor-targeted drug, rather than the mechanical or thermal action of ultrasound itself. Tumor recurrence was observed after the completion of the first treatment round; a second treatment round with the same regimen proved less effective, suggesting that drug-resistant cells were either developed or selected during the first treatment round.

Figure 1

Dropler vaporization temperature as a function of droplet size for the surface tension values of 30 mN/m and 50 mN/m.

Fig. 2

Fig. 2A. PEG-PCL nanodroplets inserted in PBS through a 18 G needle (left) or 26 G needle (right). Bubbles formed when nanoemulsion is injected through a thin needle are visualized as bright spots (indicated by arrows in the right panel); bubbles rise to the sample surface while droplets precipitate to the bottom of a test tube. Fig. 2B. PEG-PCL nanodroplets injected in the agarose gel through a 18 G (left) or 26 G (right) needle. Injection through the thin needle results in immediate formation of very bright bubbles whose size and brightness increase with time; the brightness of the droplets also increases with time suggesting a post-injection droplet-to-bubble transition.

Fig. 2

Fig. 2A. PEG-PCL nanodroplets inserted in PBS through a 18 G needle (left) or 26 G needle (right). Bubbles formed when nanoemulsion is injected through a thin needle are visualized as bright spots (indicated by arrows in the right panel); bubbles rise to the sample surface while droplets precipitate to the bottom of a test tube. Fig. 2B. PEG-PCL nanodroplets injected in the agarose gel through a 18 G (left) or 26 G (right) needle. Injection through the thin needle results in immediate formation of very bright bubbles whose size and brightness increase with time; the brightness of the droplets also increases with time suggesting a post-injection droplet-to-bubble transition.

Fig. 3

PFP/PEG-PLLA microbubbles in a plasma clot before (A) and after sonication for 1 min by 1-MHz, 3.4 W/cm² (B) and 90-kHz, 2.8 W/cm² ultrasound (C) at room temperature.

Fig. 4

Schematic illustration of drug transfer from nanodroplets through microbubbles into cells under the action of ultrasound.

Fig. 5

Photographs of a mouse bearing two ovarian carcinoma tumors (A) - immediately before and (B) - three weeks after the treatment; a mouse was treated by four systemic injections of nanodroplet-encapsulated PTX, nbGEN (20 mg/kg as PTX) given twice weekly; the right tumor was sonicated by 1-MHz CW ultrasound (nominal output power density 3.4 W/cm², exposure duration 1 min) delivered 4 hours after the injection of the drug formulation. Ultrasound was delivered through a water bag coupled to a transducer and mouse skin by Aquasonic coupling gel.

Fig. 6

Fig. 6A. Effective regression of the ovarian carcinoma tumor treated by nbGEN/ultrasound combination as described in Materials and Methods. The first photograph was taken before the start of the treatment, the second – two weeks later, i.e. immediately after the last treatment of the first treatment round. The third photograph was taken one week after the completion of the first treatment round. Fig. 6B. Normalized tumor growth/regression curve for the mouse presented in Figure 6A.

Fig. 6

Fig. 6A. Effective regression of the ovarian carcinoma tumor treated by nbGEN/ultrasound combination as described in Materials and Methods. The first photograph was taken before the start of the treatment, the second – two weeks later, i.e. immediately after the last treatment of the first treatment round. The third photograph was taken one week after the completion of the first treatment round. Fig. 6B. Normalized tumor growth/regression curve for the mouse presented in Figure 6A.

Fig. 7

Fig. 7A. Ovarian tumor growth curves for control tumors (open circles), and tumors treated by micellar PTX formulation Genexol PM (GEN, filled triangles), nanodroplet PTX formulation combined with ultrasound (nbGEN+ultrasound, filled circles), and empty nanodroplets combined with ultrasound (open diamonds). Mean values plus/minus standard errors are presented for control and nbGEN+ultrasound (N = 3). Arrows indicate days of treatment. Fig. 7B. Breast tumor growth curve for control tumor (open circles), and tumors treated with a micellar PTX formulation Genexol PM (GEN, filled triangles), and nanodroplet PTX formulation combined with ultrasound (nbGEN+ultrasound, filled circles). Mean values plus/minus standard error are presented (N = 3). Arrows indicate days of treatment.

Fig. 7

Fig. 7A. Ovarian tumor growth curves for control tumors (open circles), and tumors treated by micellar PTX formulation Genexol PM (GEN, filled triangles), nanodroplet PTX formulation combined with ultrasound (nbGEN+ultrasound, filled circles), and empty nanodroplets combined with ultrasound (open diamonds). Mean values plus/minus standard errors are presented for control and nbGEN+ultrasound (N = 3). Arrows indicate days of treatment. Fig. 7B. Breast tumor growth curve for control tumor (open circles), and tumors treated with a micellar PTX formulation Genexol PM (GEN, filled triangles), and nanodroplet PTX formulation combined with ultrasound (nbGEN+ultrasound, filled circles). Mean values plus/minus standard error are presented (N = 3). Arrows indicate days of treatment.

Fig. 8

Fig. 8A. Pancreatic tumor growth curves for control mice (open squares) and mice treated by Gemcitabine (GEM, filled squares), Genexol PM (GEN, open circles), and nanodroplet/ultrasound formulation nbGEN+GEM+ultrasound (filled triangles). Ultrasound parameters: frequency 1 MHz, nominal power density 3.4 W/cm², duration 30 s. Mean values plus/minus standard errors are presented (N = 6). Arrows indicate days of treatment. Fig. 8B. Pancreatic tumor growth curves for mice treated by Genexol PM (GEN, open circles, dashed line), Genexol PM/ultrasound (GEN+US, filled circles, dashed line), Genexol PM+GEM (open triangles, solid line), and nbGEN+GEM+ultrasound, (filled triangles, solid line). Ultrasound parameters as in 8A. Mean values plus/minus standard errors are presented (N = 6).

Fig. 8

Fig. 8A. Pancreatic tumor growth curves for control mice (open squares) and mice treated by Gemcitabine (GEM, filled squares), Genexol PM (GEN, open circles), and nanodroplet/ultrasound formulation nbGEN+GEM+ultrasound (filled triangles). Ultrasound parameters: frequency 1 MHz, nominal power density 3.4 W/cm², duration 30 s. Mean values plus/minus standard errors are presented (N = 6). Arrows indicate days of treatment. Fig. 8B. Pancreatic tumor growth curves for mice treated by Genexol PM (GEN, open circles, dashed line), Genexol PM/ultrasound (GEN+US, filled circles, dashed line), Genexol PM+GEM (open triangles, solid line), and nbGEN+GEM+ultrasound, (filled triangles, solid line). Ultrasound parameters as in 8A. Mean values plus/minus standard errors are presented (N = 6).

Fig. 9

Ultrasound images of a pancreatic tumor before (left) and 5.5 h after systemic injection of nbGEN (right).