WO2023211207A1 - Focused ultrasound treatment apparatus operating in conjunction with nanodrug carrier, ultrasound control method, and nanodrug carrier thereof - Google Patents

Abstract

Disclosed are a nanodrug carrier which delivers a nanodrug to a lesion site, a focused ultrasound treatment apparatus which operates in conjunction with the nanodrug carrier and enables the nanodrug carrier to release a nanodrug by being cavitated by ultrasonic irradiation, and an ultrasound control method. A focused ultrasound control method operating in conjunction with a nanodrug carrier according to one embodiment of the present invention comprises the steps of: obtaining an image of a lesion site; and aligning a focused ultrasound transducer on the obtained image and irradiating focused ultrasound to cavitate the nanodrug carrier at the lesion site. Through an ultrasonic control method according to one embodiment, nanodrug release characteristics of the nanodrug carrier can be effectively controlled.

Description

Focused ultrasound treatment device, ultrasonic control method, and nano-drug carrier that operate in conjunction with a nano-drug carrier

The present invention relates to a nano-drug carrier that delivers a nano-drug to a lesion site, and a focused ultrasound treatment device that operates in conjunction with a nano-drug carrier that allows the nano-drug carrier to cavitate and release the nano-drug by irradiation of ultrasound; and its ultrasonic control method.

The present invention was derived from research conducted as part of the pan-ministerial medical device research and development project of the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health and Welfare, and the Ministry of Food and Drug Safety [Project identification number: 9991006682, KMDF_PR_20200901_0009, research project Name: Commercialization and development of market-leading pancreatic cancer fusion treatment ultrasound image-guided high-intensity focused ultrasound treatment device, Research management agency: Pan-Ministerial Life Cycle Medical Device Research and Development Project Group, Contribution rate: 100%, Host research institute: IM Co., Ltd. GT, research period: 2022.3.1~2022.12.31].

Surgical incisions, radiation therapy, and chemotherapy are used to treat cancer. Among these, chemotherapy is one of the most used methods than other treatments. Conventional chemotherapy causes toxic effects on lesion sites such as tumor cells and inhibits their growth. However, chemotherapy distributes drugs randomly or widely, causing side effects in healthy tissues and biological systems. To overcome these shortcomings, a drug carrier that releases the drug only at the lesion site is needed. Drug carriers have great potential as targeted therapies that can increase treatment efficacy by increasing the accumulation of nano-sized drugs at the lesion site.

There are several types of drug carriers for anticancer drugs. Among nano-sized drug carriers, liposomes are leading the market due to their unique properties and wide range of biomedical applications. Among various nano-sized drug carriers, liposomes can stabilize drugs, increase tissue absorption, and improve bioavailability. The shape of liposomes is a spherical endoplasmic reticulum with a lipid bilayer surrounding a separate aqueous core. Because liposomes have an aqueous core and a hydrophobic lipid bilayer, they can encapsulate a wide range of drugs.

Liposomes are basically composed of a combination of various biocompatible phospholipids. And the physiological and biological properties of liposomes are fully dependent on the type of lipid that makes up the liposome. Shell properties such as surface charge, rigidity and stiffness in relation to lipid composition critically influence pharmacokinetics, biodistribution and excretion. And the properties of the shell are controlled by changing the species and ratio of lipids. However, existing liposomes are still limited in effective cancer treatment despite the progression of tumor delivery by the EPR effect, long circulation, and protection of drugs. The low therapeutic efficiency is due to the liposome's inability to release the drug in lesional tissues such as tumors. Site-specific drug release requires release in response to internal or external stimuli such as pH, temperature, enzymes, radiation, and ultrasound. Among these, the combination of nanomedicine and ultrasound is expected to overcome the limitations of nanoparticle drug delivery systems by controlling the release of drugs to desired areas in the human body.

Physical and chemical stimuli can be used to release drugs from liposomes. Chemically responsive liposomes with pH-, enzyme- or heat-dependent release mechanisms are disclosed. However, side effects may occur due to drug release in normal tissues with an environment similar to a tumor (lesion). Near infrared (NIR), radiation, and ultrasound can be means of physical stimulation to release drugs. Near-infrared rays can easily release drugs into the affected area, but their application is limited because they can only penetrate a short distance from the irradiated area. Although radiation can compensate for the shortcomings of near-infrared irradiation, continuous exposure can cause genotoxicity. On the other hand, ultrasound can achieve physical drug release that overcomes the disadvantages of near-infrared irradiation, and is the optimal method for drug release because it is safe and can reach the amount of lesions located in the body non-invasively.

Ultrasounds can induce the cavitation effect, a phenomenon in which bubbles expand to several times their resonance size and then explode during a single compression, creating high gas pressures and temperatures. These energies can cause particle destruction and are suitable for drug release from nano-sized particles. Ultrasound-induced release of liposomes can be induced using plane wave ultrasound and high-intensity focused ultrasound (HIFU). However, in order to release ultrasound-responsive drug-loaded liposomes within the desired tissue, the ultrasound release characteristics must be appropriately adjusted.

According to one embodiment, a focused ultrasound treatment device that operates in conjunction with a nano drug delivery vehicle, an ultrasonic control method thereof, and a nano drug delivery vehicle are proposed.

A focused ultrasound control method operating in conjunction with a nano drug delivery system according to an embodiment of the present invention includes the steps of acquiring an image of a lesion site; And aligning a focused ultrasound transducer with the acquired image to irradiate focused ultrasound to generate cavitation of the nano-drug carrier at the lesion site.

Additionally, the pulse repetition frequency of the focused ultrasound may be 10 to 300 Hz, the frequency of the focused ultrasound may be 20 kHz to 5 MHz, and the intensity may be 50 W/cm ² or more.

Additionally, the duty cycle of focused ultrasound may be 10% or less.

In the nano drug delivery system according to another embodiment of the present invention, cavitation occurs due to irradiation of focused ultrasound as described above, and the nano drug therein is released.

A focused ultrasound treatment device operating in conjunction with a nano drug delivery system according to another embodiment of the present invention includes an imaging transducer that acquires an image of a lesion area; And a focused ultrasound transducer aligned with the imaging transducer, wherein the focused ultrasound transducer irradiates focused ultrasound to generate cavitation of the nano-drug carrier at the lesion site visualized by the imaging transducer to deliver the nano-drug. Let it release.

And the pulse repetition frequency of the focused ultrasound may be 10 to 300 Hz, the frequency of the focused ultrasound may be 20 kHz to 5 MHz, and the intensity may be 50 W/cm ² or more.

Through the ultrasonic control method according to one embodiment, the nanodrug release characteristics of the nanodrug delivery system can be effectively controlled. In vitro doxorubicin (DOX) release using various Focused Ultra-Sound (FUS) parameters (intensity, duty cycle, pulse repetition frequency (PRF), and exposure time) to characterize the ultrasound response. As a result of the analysis, it was found that the release of DOX was proportional to the total ultrasound energy in relation to intensity and exposure time. In particular, the release of DOX was critically related to intensity and PRF. For effective DOX emission, intensity is more important than duty cycle, and PRF for cavitation is essential to generate strong acoustic wave pressure. It was found that under HIFU (High Intensity Focused Ultrasound) irradiation, the release rate of DOX was two times higher than that of common commercial drugs such as DOXIL. Therefore, it can be seen that the combination of a nano-drug delivery system and focused ultrasound according to an embodiment of the present invention showed great therapeutic efficacy in animal experiments using mice. DOX is released from liposomes accumulated in lesion tissues such as tumor tissue, killing cancer cells and having an inhibitory effect on lesions such as tumors.

And the focused ultrasound treatment device according to one embodiment can facilitate drug delivery to a specific part of the body using ultrasound images. Because various drugs can be encapsulated in liposomes, they can be applied to a variety of indications. Additionally, the use of ultrasound-responsive liposomes for targeted drug delivery can be utilized in various fields.

1A and 1B are diagrams showing the in vitro nanodrug release characteristics of a nanodrug delivery vehicle and commercial liposome DOX (DOXIL) according to an embodiment of the present invention;

2A to 2D are diagrams showing the in vitro doxorubicin (DOX) release pattern of the nanodrug delivery system according to various focused ultrasound parameters according to an embodiment of the present invention;

Figure 3 is a diagram showing a focused ultrasound treatment device according to an embodiment of the present invention, and the treatment procedure and results thereof;

Figure 4 is a diagram comparing the in vivo doxorubicin release results of a nano-drug carrier according to an embodiment of the present invention and a commercial liposome (DOXIL) under focused ultrasound irradiation using a focused ultrasound treatment device according to an embodiment of the present invention. am.

The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. The present embodiments are merely provided to ensure that the disclosure of the present invention is complete and to provide common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

In describing the embodiments of the present invention, if it is judged that a detailed description of a known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted, and the terms described below will be used in the embodiments of the present invention. These are terms defined to reflect the function of and may vary depending on the user's or operator's intention or customs. Therefore, the definition should be made based on the contents throughout this specification.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the embodiments of the present invention illustrated below may be modified into various other forms, and the scope of the present invention is not limited to the embodiments detailed below. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

First, the lipid composition of the nano drug delivery vehicle according to an embodiment of the present invention is DSPC/DSPE-PEG/cholesterol/DOPE/MSPC. Nanodrug carriers are prepared by ethanol injection followed by extrusion. Briefly, 1.50 g DSPC, 2.66 g DSPE-PEG, 2.20 g cholesterol, 9.16 g DOPE, and 0.50 g MSPC were dissolved in 62.5 mL of ethanol. The organic phase was gently heated to 60°C to dissolve the lipid components. Next, lipid-containing ethanol was injected into 437.5 mL of 250 mM ammonium sulfate solution at 250 rpm. Multilamellar vesicles (MLVs) were assembled and dispersed during ethanol injection with polycarbonate filter pore sizes ranging from 200 to 80 nm using a LIPEX® 800 mL Thermobarrel extruder (Evonik, Canada) and scaled down by successive extrusion cycles. The temperature of the vesicles was maintained at 50 °C during extrusion. The dispersion of extruded liposomes was exchanged in pH 6.5, 10% sucrose, and 10mM histidine buffer using a 12-14-kDa dialysis membrane. Ammonium sulfate was exchanged into buffer to create an ammonium gradient across the liposomal membrane. DOX was encapsulated into the aqueous phase within liposomes using a remote loading method. DOX was added to the liposome dispersion at a ratio of 1:8 to liposomes and stirred for 2 h at 37°C. DOX-loaded liposomes were diluted with buffer to a DOX concentration of 2 mg/mL and stored at 2-8 °C.

1A and 1B are diagrams showing the in vitro nanodrug release characteristics of a nanodrug delivery vehicle and commercial liposome DOX (DOXIL) according to an embodiment of the present invention.

To perform the DOX release test of the nano-drug carrier, plane wave ultrasound was irradiated with a pin-type ultrasound generator at a frequency of 24 kHz and a continuous ultrasound intensity of 92 kW/cm ² for 1 minute.

- Frequency: 24 kHz, - intensity: 92W/cm 2 , - Duty cycle: continuous wave (duty cycle 100%)

In other words, 2 mL of the liposome suspension that was ultrasonically irradiated with a pin-sonicator was loaded onto the desalting column, and then distilled water (DW) (0.5 mL) was added. An additional 4 mL of DW was added to the desalting column and collected in a cuvette to measure the absorbance of liposomal DOX at 475 nm. The reduced absorbance compared to non-irradiated liposomes indicates the amount of DOX released. The percentage of drug released was calculated using the following equation: % release = (1 - A _r /A _o ) x 100

where A _r and A _o represent the sonicated liposome suspension and original absorbance intensity, respectively.

Then, a focused ultrasound (FUS) device was used with a water bath equipped with a temperature controller and deaerator to analyze the effect of focused ultrasound on DOX release. The center frequency of the FUS transducer was 1 MHz, and the beam resolution of the focal area was 6 dB, showing a circle with a diameter of 1 mm and a circle with a length of 1 cm. To investigate HIFU-triggered DOX release, nanodrug carriers were exposed to ultrasound with various FUS parameters of intensity, duty cycle, pulse repetition frequency (PRF), and exposure time/spot. Before focused ultrasound irradiation, the nanodrug carrier was embedded in a dialysis membrane and maintained at 37°C with water degassing for 1 h. Quantification of DOX release was analyzed by measuring optical absorbance at 475 nm. The DOX released after intensive ultrasonic irradiation was purified using a desalting column, and the amount was calculated using the method described above. Commercial liposome DOX (DOXIL) was used as an ultrasound-insensitive liposome.

To compare the sound sensitivity of the nano-drug carrier according to an embodiment of the present invention and DOXIL, the two liposome solutions were exposed to low frequency continuous waves. Referring to FIG. 1A under ultrasonic irradiation with an intensity of 92W/cm ² at 24kHz, it can be seen that the nanodrug delivery system according to an embodiment of the present invention shows a DOX release rate of 58.8%. On the other hand, DOXIL only accounted for 10.2%. Therefore, it can be seen that the nano drug delivery system according to an embodiment of the present invention releases more than 5 times more DOX than DOXIL. The properties of liposomes are related to the composition and ratio of phospholipid types. Phospholipid structure determines the thermodynamic and physicochemical properties of liposomes. In the case of the nano drug delivery vehicle according to an embodiment of the present invention, the double membrane rearrangement into an inverted cone shape was achieved under ultrasound stimulation. DOPE consists of a hydrophilic head with a small hydrodynamic volume and a large hydrophobic tail, which defines a high packing parameter (PP > 1) and transforms the lamellae into an inverted hexagonal phase under sonic pressure, leading to destabilization. On the other hand, DOXIL is mainly composed of HSPC and has a cylindrical structure with equal volumes of hydrophobic chains and hydrophilic heads. This structure aligns the linear bilayer of the liposome shell and increases the stability of the liposome. Therefore, the nanodrug carrier according to one embodiment of the present invention significantly increases DOX release under ultrasonic pressure compared to DOXIL.

In high-intensity focused ultrasound (HIFU) experiments irradiated by pulse waves, the DOX release rate was similar to the release characteristics of continuous ultrasound. Referring to Figure 1a, the DOX release ratios of the nano drug delivery system and DOXIL according to an embodiment of the present invention are 48.1% and 23.3%, respectively. Ultrasound-induced destabilization of liposomes was confirmed using cryo-TEM images. The morphology of liposomes changed after ultrasound irradiation. Referring to Figure 1b, it can be seen that the frequency and thickness of DOX rods in liposomes decreased after ultrasound exposure, and the wide size distribution range also shows ultrasound-induced destabilization of liposomes.

FIGS. 2A to 2D are diagrams illustrating the in vitro doxorubicin (DOX) release pattern of a nanodrug delivery system according to various focused ultrasound parameters according to an embodiment of the present invention.

DOX release into the tumor-like lesion site was evaluated under focused ultrasound irradiation using a subcutaneous MDA-MB-231 human breast cancer xenograft model (female, balb/c nude mice, 12-14 weeks old). Nanodrug delivery vehicle and DOXIL (20 mg/kg) were injected intravenously. Focused ultrasound at an intensity of 2.0 kW/cm ² , 2.0% duty cycle, 250 Hz PRF, and 10 s/spot was irradiated after administration of each agent.

- Frequency: 0.5 ~3 MHz (preferably 1MHz), - intensity: 70.8 ~ 5644.6 W/cm 2 (2-160W) - Duty cycle: 1-80%, - pulse repetition frequency: 1- 350 Hz

To quantify the release of DOX from tumor-like lesions, fluorescence images of mice were visualized using an in vivo fluorescence imaging system. The fluorescence intensity was mainly detected at λEx/λEm = 470/560 nm. To remove autofluorescent biomolecules from the mouse body, spectral unmixing was performed and only the fluorescence intensity of DOX was observed. Referring to Figures 2A to 2D, various ultrasound conditions were used to explore the optimal ultrasound conditions for in vivo use. The results of the drug release experiment can be seen in (intensity, PRF, irradiation time and duty cycle). DOX release tends to increase as a function of ultrasound intensity. Effective release of DOX from internal liposomes requires a specific intensity threshold. In the case of a nano drug delivery system according to an embodiment of the present invention, a minimum of 2.8 kW/cm ² is required (FIG. 2a). In particular, PRF has a significant impact on the release operation of the nano drug delivery system according to an embodiment of the present invention. Referring to Figure 2b, it can be seen that the drug release rate tended to increase up to 250 Hz of PRF, but decreased above 250 Hz of PRF. PRF is an important factor related to cavitation generation. Since the frequency is related to the wave pressure of the medium, an increase in PRF causes a strong shock wave and enhances the release of DOX from the nanodrug delivery system according to one embodiment of the present invention.

Meanwhile, referring to Figure 2c, exposure time also affects the release of DOX from the nano drug delivery system according to an embodiment of the present invention. In other words, it can be seen that the emission rate increases with irradiation time. In addition, ultrasound was irradiated at various duty cycles and intensities to investigate important factors causing the release of DOX from the nano-drug delivery system according to an embodiment of the present invention. The nano drug delivery system according to one embodiment of the present invention was equally exposed to the total energy of ultrasound with a spatial peak time average (ISPTA) of 56 W/cm ² (FIG. 2d). Duty cycle and intensity are related to heat and pressure, respectively. Drug release occurred at intensities above 2.8 kW/cm ² . Frequent application of low-intensity energy has a slight effect on drug release, but temporary application of high-intensity energy leads to the release of a larger amount of drug even when the same amount of energy is irradiated. For in vivo applications, it is essential to use low-intensity ultrasound to prevent tissue damage. Ultrasound conditions for animal experiments were set at intensity 2.8 kW/cm ² , PRF 250 Hz, exposure time 10 seconds, and duty cycle 2%.

Figure 3 is a diagram showing a focused ultrasound treatment device according to an embodiment of the present invention, and the treatment procedure and results thereof.

The ultrasound-induced release of nanodrug carrier and DOX according to one embodiment of the present invention was compared using live fluorescence imaging analysis. In order to accurately expose FUS to the lesion area, such as a tumor, the lesion area is visualized three-dimensionally using ultrasound images. And the region of interest in the lesion US image for FUS exposure was determined by the number of exposure points. Then, the FUS was precisely exposed to the target area by mechanically aligning it between the FUS and the US imaging transducer. While the DOX fluorescence emission from the nanodrug carrier and DOXIL according to one embodiment of the present invention was quenched in the core of the two liposomes, the fluorescence intensity of DOX was significantly enhanced by the emission. Therefore, the nano drug delivery system and DOXIL according to an embodiment of the present invention were injected intravenously into MDA-MB-231 xenograft mice, and the lesion site was immediately irradiated with FUS.

In other words, the focused ultrasound treatment device that operates in conjunction with the nano drug delivery system includes an imaging transducer 310 that acquires an image of the lesion area and a focused ultrasound transducer 320 aligned with the imaging transducer 310, , the focused ultrasound transducer 320 emits focused ultrasound to generate cavitation of the nano-drug carrier at the lesion site visualized by the imaging transducer 310 to release the nano-drug.

Figure 4 is a diagram comparing the in vivo doxorubicin release results of a nano-drug carrier according to an embodiment of the present invention and a commercial liposome (DOXIL) under focused ultrasound irradiation using a focused ultrasound treatment device according to an embodiment of the present invention. am.

Again, Figure 4 shows DOX release after FUS exposure in an in vivo experiment. The nanodrug carrier according to one embodiment of the present invention was slightly released from the lesion area, such as a tumor, without FUS exposure. Therefore, after 1 hour of FUS irradiation, DOX was strongly released from the nano drug delivery system according to an embodiment of the present invention, and the fluorescence intensity increased in a time-dependent manner due to the release of DOX. Meanwhile, DOXIL was hardly observed at the tumor (lesion) site in both the FUS-exposed and non-exposed groups. The fluorescence intensity of DOXIL continued to quench for 6 hours. These results showed that DOXIL was fairly stable regardless of FUS exposure. On the other hand, it can be seen that the nano drug delivery vehicle according to an embodiment of the present invention effectively released DOX under FUS irradiation, and the combination of FUS and the nano drug delivery vehicle according to an embodiment of the present invention improved the anticancer effect of DOX.

Meanwhile, in one embodiment of the present invention, the description is focused on experiments on tumors and the results thereof, but the same can be applied not only to tumors but also to various lesions.

So far, the present invention has been examined focusing on its embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (9)

1. Acquiring an image of the lesion area; and

   A focused ultrasound control method operating in conjunction with a nano-drug carrier, comprising aligning a focused ultrasound transducer with the acquired image and irradiating focused ultrasound to generate cavitation of the nano-drug carrier at the lesion site. .
2. According to paragraph 1,

   A focused ultrasound control method operating in conjunction with a nano drug delivery system, wherein the pulse repetition frequency of the focused ultrasound is 10 to 300 Hz.
3. According to paragraph 1,

   A focused ultrasound control method operating in conjunction with a nano drug delivery system, wherein the frequency of the focused ultrasound is 20 kHz to 5 MHz and the intensity is 50 W/cm ² or more.
4. According to paragraph 1,

   A focused ultrasound control method operating in conjunction with a nano drug delivery system, wherein the duty cycle of the focused ultrasound is 10% or less.
5. A nano-drug delivery system characterized in that cavitation is generated by irradiation of focused ultrasound according to any one of claims 1 to 4, thereby releasing the nano-drug therein.
6. An imaging transducer that acquires images of the lesion area; and

   Comprising a focused ultrasound transducer aligned with the imaging transducer,

   The focused ultrasound transducer irradiates focused ultrasound to generate cavitation of the nano-drug carrier at the lesion site visualized by the imaging transducer to release the nano-drug, and operates in conjunction with the nano-drug carrier. Ultrasound treatment device.
7. According to clause 6,

   A focused ultrasound treatment device that operates in conjunction with a nano drug delivery system, wherein the pulse repetition frequency of the focused ultrasound is 10 to 300 Hz.
8. According to clause 6,

   A focused ultrasound treatment device that operates in conjunction with a nano drug delivery system, wherein the frequency of the focused ultrasound is 20 kHz to 5 MHz and the intensity is 50 W/cm ² or more.
9. According to clause 6,

   A focused ultrasound treatment device that operates in conjunction with a nano drug delivery system, wherein the duty cycle of the focused ultrasound is 10% or less.

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