Light-Activated Doxorubicin-Encapsulated Perfluorocarbon Nanodroplets For On-Demand Drug Delivery in an in vitro Angiogenesis Model: Comparison Between Perfluoropentane and Perfluorohexane

Abstract

Phase-transition perfluorocarbon (PFC) nanodroplets have been developed for on-demand drug delivery carriers with external triggers such as ultrasound or laser irradiation techniques. Although various perfluorocarbons, including perfluoropentane (C₅F₁₂) and perfluorohexane (C₆F₁₄), have been investigated for their theranostic use, comparison of the phase-transition efficiency, the drug delivery efficacy by light activation, and physical properties of the PFC nanodroplets have not been reported. We have synthesized gold nanorod-coated doxorubicin-encapsulated perfluorocarbon nanodroplets using perfluoropentane and perfluorohexane as light-activated on-demand drug delivery carriers, called PF5 and PF6, respectively. When gold nanorods on the perfluorocarbon nanodroplets resonate with a laser wavelength, plasmonic heat generated on the gold nanorods vaporizes the nanodroplets to gas bubbles (phase-transition), and releases the encapsulated drug from the nanodroplet core.

Overall, the nanodroplet size, drug encapsulation efficiency, number density, and cytotoxicity were similar between PF5 and PF6. However, the long-term stability against passive phase-transition or coalescence in physiological conditions and the phase-transition efficiency were different from each other. PF6 was better in long-term stability but showed lower phase-transition than PF5. The lower phase-transition of PF6 might have led to lower drug delivery efficiency compared to PF5. This is probably because PF6 has higher temperature thresholds required for phase-transition due to its higher boiling point. The study demonstrated feasibility of the light-activated nanodroplets for on-demand targeted nanotherapy, which suppresses the development of angiogenesis.

Graphical Abstract

1. Introduction

Angiogenesis is an essential hallmark of cancer. Tumor vessels sprout for sustained neoplastic growth and dissemination of tumor. ^1–3 Therapeutic local delivery to tumor angiogenesis shows great potential for preventing cancer progression.⁴ Targeting angiogenesis can be an effective approach to prevent the development of new blood vessels; ⁵ thus, it can prevent the development of tumors and can serve as a cancer therapy.⁶ Nanotherapeutics using nanoparticles enhance therapeutic efficacy and avoid adverse side effects of conventional chemotherapy, via local delivery strategy.⁷

Phase-transition perfluorocarbon (PFC) nanodroplets have been developed and extensively investigated for contrast-enhanced ultrasound imaging, in which the liquid in the core of nanodroplets vaporizes to gas phase upon ultrasound exposure.^8–10 Drug-contained PFC nanodroplets also have been developed for real-time imaging-guided on-demand drug delivery.^{11, 12} The PFC nanodroplets are stable in aqueous phase until they are triggered to vaporization for drug release. Combined with plasmonic materials, the nanodroplets are activated by laser or light and release drug in a precise controlled manner targeting lesions to minimize adverse effects to surrounding tissues. By choosing the duration, energy, or beam area, the drug release can be intricately controlled compared to other methods, including local heating.^{13, 14} In addition, a laser can be used for gaseous enclosures in the human body such as the lungs and pressure-sensitive organs such as the eyes, whereas ultrasound beams cannot effectively be used in those areas.¹⁵ Several studies showed the feasibility of delivering drugs upon laser irradiation in vivo using phase-transition nanodroplets for cancer therapy.^{16, 17}

Among various perfluorocarbon examples, perfluorobutane (C₄F₁₀) and perfluoropentane (C₅F₁₂) have been widely studied because of low activation threshold due to their low boiling temperatures, −1.7 and 28 °C (bulk liquid), ¹⁸ respectively.^{8, 19–22} Recently, perfluorohexane (C₆F₁₄) has been used for ultrasound contrast-agents in a nanodroplet form because of the stability against passive vaporization in physiological conditions partly due to its high boiling point at 56 °C.²³ Also perfluorohexane nanodroplets have been developed for a drug delivery carrier because of the excellent stability in the body. To trigger phase-transition of perfluorohexane nanodroplets for drug-release, it requires higher energy due to its higher boing point compared to perfluoropentane or perfluorobutane.²⁴ However, safety of using the high laser intensity to surrounding cells is often not reported. Most studies do not differentiate the effect of drug delivery from the effect of heat, so-called photothermal therapy, on killing cancer cells, when treating with laser. In contrast, this study focuses on suppressing angiogenesis by delivering drug activated by laser, not by heating, considering minimization of collateral damage to surrounding tissues.

We previously developed gold-nanorod-coated perfluorocarbon nanodroplets as a light-activated drug delivery system using perfluoropentane and perfluorohexane.^{24, 25} (Figure 1). In this study, the perfluoropentane nanodroplets (PF5) and the perfluorohexane nanodroplets (PF6) will be compared for stability, yield of production, encapsulation efficiency, phase-transition efficiency, drug delivery efficiency, and cytotoxicity using an in vitro angiogenesis model. We have already demonstrated phase-transition phenomena of PF5 and PF6 and explained the mechanism of the phase-transition, thermal energy needed, and estimated temperature rise due to the photothermal process via integrated experimental and theoretical approach.²⁴ Briefly, the temperature threshold of phase-transition for PF6 was higher than PF5 (> 80 and > 60 °C, respectively). Here we focus on comparing properties in biomedical applications, and evaluating the feasibility of using these light-activated nanodroplets for cancer therapy by suppressing angiogenic vessels growth.

Figure 1.

Schematics of phase-transition of a gold nanorod-coated perfluorocarbon nanodroplet to a micron-sized bubble by pulsed laser on an in vitro angiogenesis model.

2. Materials and Methods

2.1. Nanodroplet Synthesis, Materials, and Preparation

The nanodroplets were obtained via a double emulsion procedure as described in our previous study ²⁵. Briefly, to prepare the first emulsion, doxorubicin hydrochloride (MP Biomedicals, Solon, OH) solution (0.3 mL, 10mg/mL) was dispersed in 1.55 mL liquid perfluoropentane (C₅F₁₂) or perfluorohexane (C₆F₁₄) (Synquest Laboratories, Alachua, FL) with 0.15 mL of Krytox 157 FSL (DuPont) as a surfactant by a tip sonicator at pulse-mode (10s on, 20s off at 20% amp.) for 1 min in an ice bath. The first emulsion was added to a liposome aqueous dispersion (described in the next paragraph) at a ratio of 1:2 (v/v, emulsion/liposome) and sonicated by probe sonication (10s on, 20s off at 20% amp. for 1 min). The resulting suspension was centrifuged at 4,000 g-force for 3 min to collect a pellet of nanodroplets. Gold nanorods were attached on the nanodroplet surface by mixing an aqueous gold-nanorod solution (Nanopartz, Inc. Loveland, CO) via the electrostatic interaction. Then the gold nanorod-coated nanodroplet suspension was washed 3 times via centrifugation (4,000 g-force, 3 min) to synthesize the light-activated doxorubicin (Dox)-encapsulated perfluorocarbon nanodroplets, dispersed in deionized (DI) water. The nanodroplets synthesized with perfluoropentane and perfluorohexane for the perfluorocarbon core are PF5 and PF6, respectively.

The liposomes were prepared prior to the synthesis of nanodroplets. Briefly, stearylamine (Tokyo Chemical Industry Co., Portland, OR), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000 (DSPE-PEG 5000), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids, Inc., Alabaster, AL) were mixed at a molar ratio of 50: 15: 35 (stearylamine: DSPE-PEG 5000: DSPC) in chloroform and dried in a chemical hood overnight to form a thin film. The dry lipid film was hydrated with 2 mL of DI water and sonicated via probe sonication (10 min) to form the liposomes.

2.2. Characterization of Nanodroplets

2.2.1. Optical Imaging

The fluorescence images of the Dox which is encapsulated in the nanodroplets were obtained via a fluorescence microscope (Nikon, Inc., Tokyo, Japan). The existence of gold-nanorods on the nanodroplet surface were observed using an enhanced darkfield hyperspectral microscope (CytoViva, Inc., Auburn, AL).

2.2.2. Transmission Electron Microscopy (TEM)

The morphology and size of the gold nanorod-coated perfluorocarbon nanodroplets were characterized by a transmission electron microscope (JEOL, JEM-1230). Briefly, the nanodroplet suspension was diluted 5× with DI water. A drop of the diluted sample (~5 µL) was put on the parafilm and a 400-mesh copper grid (Electron Microscopy Sciences Inc., Hatfield, PA) was carefully placed on the top of the drop with the carbon-coated side down to absorb the sample. After 1 min, the grid was removed from the sample and the excessive liquid was blotted away with a filter paper. Then a drop of 2% uranyl acetate (w/v in DI water) was placed on the parafilm. The grid with the sample was carefully put on the drop of uranyl acetate for 10~30 seconds to stain. The grid was air dried for 10 min prior to the TEM imaging.

2.2.3. Dynamic light scattering (DLS)

Dynamic light scattering (NanoBrook Omni, Brookhaven Instruments Co., Holtsville, NY) was used to determine the hydrodynamic diameter of the nanodroplets.

2.3. Stability of Nanodroplets

The stability of nanodroplets in physiological conditions against dissolution, aggregation, or passive phase-transition was monitored for 40 days using DLS. The nanodroplets dispersed in medium were divided into aliquots (200 µL each) and stored in an incubator (Model 3110, Forma Scientific, Inc., Mariette, OH) at 37° C. The samples were diluted 40× in DI water when measured by DLS in a cuvette.

2.4. Yield of Production of Nanodroplets

The yield of nanodroplet production was determined by the number density after synthesis. The number density was determined by analyzing optical images (Carl Zeiss, Axio Observer A1, Oberkochen, Germany) using a hemocytometer. The nanodroplet suspensions were diluted 500× and treated with trypan blue (Corning Inc., Corning, NY) to enhance contrast compared to the background. The number density of nanodroplets was calculated by the formula below.

$$Number\hspace{0.17em}density=\frac{Average\hspace{0.17em}nanodroplets\hspace{0.17em}per\hspace{0.17em}square\hspace{0.17em}(n=4)\hspace{0.17em}\times \hspace{0.17em}\hspace{0.17em}Dilution\hspace{0.17em}factor}{Volume\hspace{0.17em}of\hspace{0.17em}one\hspace{0.17em}square}$$

where the dilution factor was at 500× and the volume of the square was 6.25 nL. The measurement was conducted in triplicate.

2.5. Drug Encapsulation Efficiency

A calibration curve of Dox dissolved in water/acetonitrile (ACN) solution (1:1, v/v) was generated by measuring UV-Vis optical density (OD) of Dox at 480 nm at a serial of concentrations from 0.01 µg/mL to 100 µg/mL with a UV-vis spectrometer (SpectraMax, Molecular Devices, LLC). The calibration curve is in the Supporting Information.

To determine the Dox concentration in the nanodroplets, 100 µL of nanodroplet suspension was mixed with the equal volume of an ACN solution to dissolve Dox in the water/ACN solution (1:1). ACN allows to rupture the nanodroplets and release the Dox to the solution. Then the UV-Vis OD of the mixture at 480 nm was measured to determine Dox concentration based on the calibration curve. The encapsulation efficiency was determined by the formula below:

$$Encapsulation\hspace{0.17em}Efficency\%\hspace{0.17em}=\frac{Dox\hspace{0.17em}concentration\hspace{0.17em}measured}{Theoretical\hspace{0.17em}Dox\hspace{0.17em}concentration}\hspace{0.17em}\hspace{0.17em}\times \hspace{0.17em}\hspace{0.17em}100.$$

The theoretical Dox concentration in synthesized nanoparticles was 60 µg/mL.

2.6. Laser Activation of Nanodroplets (Phase-transition)

A picosecond pulsed laser (WedgeHF, RPMC Lasers Inc.) at 1064 nm, 1.08 W average power, 500 ps, and 10 kHz pulse repetition frequency was used throughout the phase-transition and drug release experiments except for the phase-transition observation experiment where an optical microscope was used. For the optical observation experiment, a Mai Tai (Spectra Physics) femtosecond laser at 980 nm, 0.08 W average power (measured), 100 fs, and 80 MHz pulse repetition frequency was used.

The number of nanodroplets were quantitatively determined by light attenuation of the nanodroplet suspensions at different dilution factors using a UV-vis spectrometer at 633 nm. Combined with the number density data counted from a hemocytometer as described in previous section 2.4, the light attenuation measurement values were linearly proportional to the number density (Supporting information).

The phase-transition efficiency of the nanodroplets was determined by the following formula:

$$Phase\hspace{0.17em}transition\hspace{0.17em}efficiency\hspace{0.17em}=\hspace{0.17em}\frac{number\hspace{0.17em}of\hspace{0.17em}nanodroplets\hspace{0.17em}phase-transitioned\hspace{0.17em}after\hspace{0.17em}laser\hspace{0.17em}irradiation}{total\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}nanodroplets\hspace{0.17em}before\hspace{0.17em}laser\hspace{0.17em}irradiation}.$$

2.7. In Vitro Angiogenesis Model

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Bioscience. They were cultured in EBM™−2 Basal Medium (Lonza), containing 10% fetal bovine serum and the EGM™−2 SingleQuots™ Supplements (Lonza) required for growth of endothelial cells at 37° C, 5% carbon dioxide. For formation of tubular structure, the 96-well plate was coated with Geltrex™ Matrix (40µL/well) and incubated at 37°C for 30 min. HUVECs (15,000 cells, passage number 9) in 100 µl EGM™−2 were seeded onto the matrix-coated well. Angiogenic vessels formed after 16 hours of incubation were used for drug delivery efficacy examination. Tubulogenesis was confirmed using an inverted microscope (Axio Observer A1, Carl Zeiss, Germany).

2.8. In Vitro Drug Delivery Efficiency

The nanodroplets suspended in EGM™−2 medium (100 µL) were added to the angiogenesis model created in 96-well plate by replacing existing medium in each well. For the laser group, the picosecond laser (WedgeHF, RPMC Lasers Inc.) was irradiated into each well for 20 s at two different spots (40 s total). The cell mortality was analyzed after 24 hr incubation using SYTOX Green (Life Technologies, Carlsbad, CA) and Hoechst 33342 (Life Technologies) following the product instructions, to demonstrate dead cells and total cells, respectively. The cell mortality was calculated as the following: The number of dead cells (Sytox Green)/the number of total cells (Hoechst 33342) x 100%. The numbers of the dead/total cells in optical images were manually counted. To follow conventional colors, red color was used for dead cells stained by Sytox Green when demonstrating the results. Six conditions were tested, including Dox nanodroplets with/without laser irradiation, empty nanodroplets with laser irradiation, cell culture medium with laser irradiation, cell culture medium (negative control) and a Dox solution at 0.6 µg/mL (positive control, free Dox). Dox concentrations in the Dox nanodroplets suspended in the medium matched the free Dox at 0.6 µg/mL. Empty nanodroplets were produced by substituting Dox solution with a DI (DI) water in the synthesis. The nanodroplet number density matched throughout each well based on dilution factor (~10⁸/mL).

2.9. Temperature change measurement

The medium temperature of the in vitro condition in Section 2.8 was measured to confirm any collateral damage to the cells by heating during laser irradiation. Briefly, the EGM™−2 medium, PF5, and PF6 suspended in the medium (100 µL each) were added to separate wells in a 96-well plate in a 37°C water bath. After the temperature of the system was stabilized at 37 °C, the picosecond laser was irradiated into each well for 20 s at two different spots (40 s total). Meanwhile, a FLIR T640 camera (FLIR System Inc., Wilsonville, OR) was set at spot-mode and targeted at the center of the well to measure the temperature in real-time until the temperature returned to 37°C. Dox concentration in the nanodroplet suspensions was approx. 0.6 µg/mL. The nanodroplet number density matched throughout each well (~10⁸/mL).

2.10. Statistical Analysis

For statistical analysis, each condition was triplicated and three random areas of each well were used for imaging analysis. The cell viability was determined by the ratio of dead cells to total cells. A two-sample t-test was used to determine p-values. A statistically significant difference between two samples was determined when the p-value is less than 0.05.

3. Results

3.1. Nanodroplets Characterization

Detailed characterization of the nanodroplets is described in our previous publications.^{11, 24} Briefly, Dox, which is naturally fluorescent, was observed only in the core of both PF5 and PF6 (red color in Figure 2A). This also visually proves Dox in W1 phase with a double emulsion structure of W1/O/W2. The size of both PF5 and PF6 measured by optical images and dynamic light scattering ranged 300 ~ 500 nm in diameter (Figures 2A and 2B). Both PF5 and PF6 had a narrow size distribution when they were freshly prepared at Day 0 (Figure 2B). The PF5 had an average diameter at ~465 nm, which is relatively bigger than PF6 at ~350 nm. This result was consistent with optical observations obtained by TEM and optical microscopes. The peak near 100 nm may be due to debris from phase-transitioned nanodroplets or free gold nanorods.

Figure 2.

Characterization of the PF5 and PF6: (A) Fluorescence optical image (60×); (B) Representative dynamic light scattering data: size distributions of the nanodroplets at Day 0; (C) TEM image (red arrow illustrates gold nanorod); (D) Enhanced dark-filed image.

Gold nanorods on both the PF5 and PF6 were observed via TEM imaging and enhanced dark-field imaging in Figure 2C and 2D, respectively. The arrow in Figure 2C indicates the gold nanorod attached on the nanodroplet (PF5) with a dimension of 10 × 60 nm². The blue color from the surface of the nanodroplets in Figure 2D indicates presence of gold nanorods. Nanodroplets without gold nanorods on the surface show orange color in comparison.¹¹

3.2. Particle Stability Comparison

The average diameter of PF6 was 350 ~ 400 nm with no significant changes while PF5 showed a noticeable fluctuation in the diameter upon the storage in the medium for 40 days at 37°C. After Day 24, PF5 tended to be stable at a reduced average diameter of ~360 nm. The results may suggest that unstable nanodroplets in PF5 undergo merging and phase-transition. The peaks above 1,500 nm in the size distribution of PF5 in Figure 3B demonstrated the existence of large aggregates or microbubbles at Day 40. The peak near 100 nm may be due to debris from phase-transitioned nanodroplets or free gold nanorods. Similarly, peaks at 875 nm and 100 nm for PF6 indicate instability at Day 40 (Figure 3C). Nevertheless, the dominant peaks for both PF5 and PF6 were close to the original size at Day 0, suggesting that the nanodroplets remained stable at least for 40 days in physiological conditions.

Figure 3.

(A) Size stability of PF5 and PF6 in medium over 40 days at 37°C monitored by DLS; Size distributions on Day 40 after stored at 37°C by DLS (n = 3) of (B) PF5 and (C) PF6.

3.3. Yield, Drug Encapsulation Efficiency & Phase-Transition Efficiency

The phase-transition process of PF5 and PF6 was demonstrated utilizing an optical microscope equipped with a femtosecond laser (Figure 4). The perfluorocarbon cores of the nanodroplets converted into microbubbles immediately upon the laser irradiation. After phase-transition, bubbles started to increase in size and merge with adjacent bubbles over time.

Figure 4.

Representative optical images of phase-transition of perfluorocarbon nanodroplets (PF5) by femtosecond laser.

The yield of the particle number density for freshly prepared PF5 and PF6 was 3.11 × 10⁹ and 2.17 × 10⁹ /mL, respectively with no significant difference (p = 0.26) (Table 1). The encapsulation efficiencies of Dox in PF5 and PF6 were also similar to each other at 9.63 ± 0.76% and 8.40 ± 0.87%, respectively, with no significant difference (p = 0.21). By measuring the Dox concentration in each sample, the average Dox concentration per particle was estimated for both PF5 and PF6. However, upon laser irradiation, the PF5 showed a phase-transition efficiency of 25.10 ± 0.57%, which is significantly higher than that of PF6, 11.85 ± 1.02% (p = 0.0007).

Table 1.

The comparison of PF5 and PF6 nanodroplets on the average yield, the encapsulation efficiency and the phase-transition efficiency.

Nanodroplet Sample	Number Density (×109/mL) *	Drug Encapsulation Efficiency**	Avg. Dox per Particle (×10−9 μg/particle) ***	Phase-transition Efficiency (%) ****
PF5	3.11 ± 0.40	9.63 ± 0.76%	1.88 ± 0.18	25.10 ± 0.57%
PF6	2.17 ± 0.92	8.40 ± 0.87%	2.74 ± 1.10	11.85 ± 1.02%

^*p = 0.26

^**p = 0.21

^***p = 0.33

^****p = 0.0007

3.4. In Vitro Drug Delivery Efficacy

Figure 5 represents the process of treating the angiogenic tubes with the nanodroplets and subsequently triggering them with a laser for drug release. The left image shows angiogenesis formed after 16-hr incubation. The middle image shows the nanodroplets (arrows) added to the angiogenesis model. The image of microbubbles (arrow heads) were captured right after laser irradiation for 20 s at two different spots (the right image). As shown in Figure 4, the microbubbles might have been merged, showing 10~50 μm in size. The center of the well appears darker probably due to meniscus effect. A dark spot always shows in the center as long as it is filled with liquid (without cell or gel matrix) in a 96 well plate, when observed by 4x objective lens.

Figure 5.

Representative images showing the process of in vitro drug delivery experiment. Left: angiogenesis before adding the nanodroplets; Middle: after adding the nanodroplets (arrows) and before laser irradiation; Right: after laser irradiation, micro-bubbles (arrow heads) formed from the phase-transition process.

In Figure 6, PF5 nanodroplets after laser activation (Condition #1) showed a cytotoxicity of 50.6 ± 3.9%, which is significantly different compared to a control, 23.2 ± 5.9% (Condition #6, medium only) (p = 0.002). The results suggested Dox was released by laser and delivered to the cells. The cytotoxicity of empty nanodroplets (without Dox) with laser (Condition #2, 11.7 ± 5.0%) was similar to Condition #6, demonstrating that there is no cytotoxicity from either nanodroplets or the phase-transition process. Condition #4, laser only without nanodroplets, appeared to be harmless to the cells with a cytotoxicity of 11.3 ± 5.0%. Dox-encapsulated PF5 nanodroplets without laser (Condition #3) had a cytotoxicity of 34.7% ± 4.0% with no significant difference from Condition #6 (p > 0.05).

Figure 6.

(A) Cell mortality (%) data calculated based on the number of total and dead cells from fluorescence images (*p < 0.05, **p < 0.005 compared to control group, Condition #6, Medium only). (B) Representative images of the bars indicated in (A) left: fluorescence images (total cells, green; dead cells, red); right: bright-field images.

For the positive control group (Condition #5) where cells were incubated with free Dox in the medium, the cytotoxicity was 88.3% ± 2.1%, which also showed p values < 0.005 compared to all other conditions, Conditions #1~#5.

For the PF6, the trend of the mortalities was similar to that of PF5. However, it is noted that the PF6 had a lower cytotoxicity of 41.0% ± 3.1% compared to 50.6% ± 3.9% of PF5 (p < 0.05) when irradiated by the laser (Condition #1). The results may indicate that the number of PF6 activated was less than the PF5, which is consistent with the phase-transition efficiency data in the previous section. In addition, the PF6 without laser (Condition #3) showed a lower cell cytotoxicity than the PF5 (p = 0.04). This could be due to the better stability against dissolution during the incubation, as shown in Figure 2.

3.6. Surrounding Temperature Measurement

The surrounding temperature of the in vitro conditions in the previous section was measured to confirm the effect of overall temperature on the cell mortality results. By the end of the irradiation, the temperatures of all the three conditions (medium only, PF5, and PF6) increased by 4.10 ± 0.28, 4.50 ± 0.22 and 4.32 ± 0.24°C respectively (p > 0.05 for all pairwise combinations). When the irradiation ended, the temperatures decreased dramatically within 10 seconds and eventually stabilized at 37°C after 90 seconds. There was no significant difference between the three groups on the surrounding temperature changes. The results suggest that PF5 and PF6 do not cause additional heat which would be detrimental to the cells. Also the mechanism of drug release is due to local plasmonic heat generated from the gold nanorod on the nanodroplet surface, not the entire temperature rise of the in vitro well.

4. Discussion

The light-activated perfluorocarbon nanodroplets made of perfluoropentane and perfluorohexane, PF5 and PF6, respectively, showed similar physical properties with respect to the size, drug encapsulation efficiency and gold nanorod-coating. However, PF6 showed better long-term stability in the medium at 37 °C compared to PF5 although both PF5 and PF6 did not change in size for about 7 days. PF5 started to show merging and debris based on the size distribution data from DLS measurement from Day 7.

Considering the Laplace-Young surface pressure, the boiling temperatures of PF5 and PF6 calculated by Antoine equation are 31.6 °C and 61.0 °C, respectively (r = 200 nm).²⁴ Based on the calculation, all the PF5 nanodroplets should undergo phase-transition immediately when stored at 37°C. The stability of PF5 for at least 7 days may come from metastability of superheated fluid,²⁶ as opposed to PF6 which is thermodynamically stable.

When the in vitro angiogenesis model was treated by the laser, the temperature increased to near 41 °C and returned to 37 °C within 90 seconds, with or without the nanodroplets. The previous study showed that PF5 and PF6 had similar stability against phase-transition and merging even at 40 °C for a short period of time (5 min).²⁴ Thus, the results suggest that (1) the phase-transition was due to triggering the metastable nanodroplets by plasmonic heating of the nanodroplet surface, not by heating the surrounding medium; and (2) the cell mortality results were due to drug delivery from the nanodroplets, not by any collateral heating damage.

We observed that the overall phase-transition efficiency of PF6 was lower than that of PF5 (11.9% vs. 25.1%, Table 1). This could be due to the temperature threshold to overcome phase-transition. Our previous study has shown that the temperature values required for phase-transition for PF5 and PF6 are > 60 °C, and > 80 °C, respectively.²⁴ The lower phase-transition of PF6 may also be due to recondensation of PF6 nanodroplets, according to studies recently reported.^26–27 Perfluorohexane (C₆F₁₄) droplets have been used for contrast-enhanced ultrasound imaging because of their ability to vaporize and recondense, which is beneficial for long-term use.^{23, 27, 28} We did not focus on the recondensation process in this study because drug delivery is determined by the final amount of nanodroplets phase-transitioned. In future work, we can investigate the recondensation process focusing on individual nanodroplets.

We previously tested whether bubbles are induced by laser either with gold nanorod alone or bare nanodroplets alone (without gold nanorod coating).¹¹ In the same experimental setting, bubbles were not detected by both ultrasound imaging and optical imaging. Nano-sized bubbles smaller than the detection limit may have formed. However, we believe that laser alone did not affect the cell mortality results because our previous study has shown that Sytox Green did not penetrate the cell walls in the presence of cavitation effect of bubbles.²⁹ Thus, Sytox uptake is mainly due to cell death by Dox, not by other effects, such as phase-transition process. However, in order to conclude the effect of phase-transition and predict drug delivery efficiency by correlating with the phase-transition efficiency data, we may need to add various laser conditions.

Lastly, the lower phase-transition efficiency of PF6 might have caused the lower cell cytotoxicity compared to PF5 (50.6% vs. 41.0%). The laser did not activate the whole nanodroplets added to the cells because two spots in each well was irradiated, instead of the whole area to cover all the nanodroplets. Therefore, a portion of the drug was released by the laser in vitro experiment, and condition #1 does not necessarily have to be greater than the sum of condition #2 and condition #3. Theoretically, three and four times of repetitive treatment will reach the maximum cell cytotoxicity (88% with free Dox) with PF5 and PF6, respectively, considering 20% of cell mortality with medium only.

5. Conclusions

In conclusion, because of different temperature thresholds to trigger phase-transition for drug delivery between PF5 and PF6, it is important to note that different laser parameters may be required to obtain the same drug delivery results. At the same time, it is also critical to determine optimal laser parameters to avoid collateral damage due to laser heating. For long-term use in the body, PF6 will be better than PF5 because of the better stability. However, the phase-transition efficiency of PF5 was better probably due to the lower temperature threshold. Lastly, the study demonstrated feasibility of light-activated nanodroplets for on-demand targeted nanotherapy, which suppresses the development of angiogenesis.

Figure 7.

(A) A representative images of temperature change captured with a FLIR camera. The temperature reading is at the target spot (arrow). (B) The temperature changes upon and after the laser irradiation for medium only, PF5 and PF6 in the medium. Red arrows indicate the start and the end of the irradiation.

Highlights.

• Perfluorocarbon nanodroplets delivered drug on demand by laser activation.

• In vitro angiogenic vessels were killed by drug released, not by photothermal heating.

• We compared various properties of PF5 and PF6 for biomedical applications.

• PF6 has better stability than PF5 in physiological conditions after one week of incubation.

• PF6 has less phase-transition efficiency than PF5, which might have led to lower drug delivery efficiency.