WO2000002590A1

WO2000002590A1 - Radiation and nanoparticles for enhancement of drug delivery in solid tumors

Info

Publication number: WO2000002590A1
Application number: PCT/US1999/015025
Authority: WO
Inventors: Rinat O. Esenaliev
Original assignee: The Board Of Regents Of The University Of Texas System
Priority date: 1998-07-09
Filing date: 1999-07-01
Publication date: 2000-01-20
Also published as: US6165440A; AU4854599A

Abstract

The present invention discloses a method/system utilizing interaction of electromagnetic pulses or ultrasonic radiation with nano- and microparticles for enhancement of drug delivery in solid tumors. The particles can be attached to antibodies directed against antigens in tumor vasculature and selectively delivered to tumor blood vessel walls. Cavitation induced by ultrasonic waves or local heating of the particles by pulsed electromagnetic radiation results in perforation of tumor blood vessels, microconvection in the interstitium, and perforation of cancer cell membrane, and therefore, provides enhanced delivery of macromolecular therapeutic agents from blood into cancer cells with minimal thermal and mechanical damage to normal tissues.

Description

RADIATION AND NANOPARTICLES FOR ENHANCEMENT OF DRUG DELIVERY IN SOLID TUMORS

BACKGROUND OF THE INVENTION

Cross-reference to Related Application

This patent application claims benefit of non-provisional

patent application U.S. Serial number 09/112,491 , filed July 9, 1998.

Field of the Invention

The present invention relates generally to the fields of

immunology and cancer therapy. More specifically, the present

invention relates to a system utilizing interaction of electromagnetic

pulses or ultrasonic radiation with nanoparticles to enhance anti-

cancer drug delivery in solid tumors and uses of such a system. Description of the Related Art

Many promising therapeutic agents have been proposed

for cancer therapy for the past two decades. Their potential is proven

in numerous preclinical studies. However, limited success has b een

achieved in solid tumor therapy. The presence of physiological

barriers to drug delivery in tumors substantially limits efficacy of th e

anti-cancer drugs. To penetrate into cancer cells in a solid tumor,

therapeutic agents have to pass through blood vessel wall, interstitial

space, and cancer cell membrane. Penetration of anti-cancer drug s

through these physiological barriers is poor especially for the most

promising macromolecular therapeutic agents such as monoclonal

antibodies, cytokines, antisense oligonucleotides, and gene-targeting

vectors.

Methods have been reported for delivery of anti-cancer

drugs with low molecular weight in solid tumors. Many of them are

based on selective delivery of particles loaded with the drugs i n

tumors. It has been demonstrated that particles coated with a

surfactant have prolonged circulation time and selectively accumulate

in tumors because of increased leakage of tumor vasculature in

comparison with the normal one. These long-circulating particles

avoid rapid clearance by reticuloendothelial system. This approach is

referred to as "passive" delivery of particles in tumors. The "active" delivery is based on attachment of long-

circulating particles to antibodies directed against antigens in tumor

vasculature. Results of studies on animals bearing tumors derived

from human cancer cells demonstrate feasibility of active delivery of

anti-cancer drugs to tumor vasculature. These antibodies and short

peptide sequences can be used for targeting anti-cancer drugs in

patients .

The prior art is deficient in the lack of effective means of

enhancing the delivery of anti-cancer drugs (especially

macromolecular ones) from tumor blood vessels into cancer cells with

minimal damage to normal tissues. The present invention fulfills this

long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method or system of

utilizing the interaction of electromagnetic pulses or ultrasonic

radiation with nanoparticles and microparticles for enhancement of

drug delivery in solid tumors. The particles can be attached to

antibodies directed against antigens in tumor vasculature an d

selectively delivered to tumor blood vessel walls. Cavitation induced by ultrasonic waves or local heating of the particles by pulsed

electromagnetic radiation results in perforation of tumor blood

vessels, microconvection in the interstitium, and perforation of cancer

cell membrane. This method provides enhanced delivery of

macromolecular therapeutic agents from the blood into cancer cells

with minimal thermal and mechanical damage to normal tissues.

In one embodiment of the present invention, there is

provided a method of enhancing anti-cancer drug delivery in a solid

tumor, comprising the steps of administrating at least one anti-cancer

drug to the tumor; injecting nanoparticles or microparticles to th e

tumor intravenously; and irradiating the tumor with radiation.

Generally, the anti-cancer drug is selected from the group

consisting of a monoclonal antibody, a cytokine, an antisense

oligonucleotide, a gene-targeting vector and any other macromolecular

therapeutic agent. Generally, the tumor occurs in the organ selected

from the group consisting of breast, lung, brain, liver, skin, kidney, GI

organ, prostate, bladder, gynecological organ and any other hollow

organ.

Preferably, the nanoparticles or microparticles are long-

circulating particles with or without antibody coating, wherein the

antibody is directed against tumor vasculature. The nanoparticles or

microparticles can be metal particles, carbon particles, graphite particles, polymer particles loaded with an absorbing dye, liquid

particles loaded with an absorbing dye or porous particles having gas-

filled pores. The nanoparticle has a diameter from about 0.1 nm to

about 7000 nm.

Preferably, the radiation is optical pulsed radiation

generated from a laser or non-laser source. Specifically, the optical

radiation is in the spectral range from 0.2 μm to 2 μm and delivered

through the skin surface or via optical fibers inserted in a needle or

endoscopes to the tumor.

Preferably, the radiation is ultrasonic radiation generated

from an ultrasonic transducer. Specifically, the ultrasonic radiation is

in the frequency range from 20 to 500 kHz and delivered through the

skin surface to the tumor.

In another embodiment of the present invention, there is

provided a system for enhancement of anti-cancer drug delivery in a

solid tumor, comprising a source of radiation; an electronic system or

means for monitoring of the radiation; a system or means for delivery

of the radiation to the tumor; nanoparticles or microparticles

absorbing the radiation; an injection system or means for

administration of the anti-cancer drug and the nanoparticles or

microparticles in tumor blood.

In still another embodiment of the present invention, there is provided a method of treating a solid tumor, comprising the

steps of injecting nanoparticles or microparticles to the tumor

intravenously and irradiating the tumor with radiation.

Other and further aspects, features, and advantages of th e

present invention will be apparent from the following description of

the presently preferred embodiments of the invention given for th e

purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features ,

advantages and objects of the invention, as well as others which will

become clear, are attained and can be understood in detail, more

particular descriptions of the invention briefly summarized above

may be had by reference to certain embodiments thereof which are

illustrated in the appended drawings. These drawings form a part of

the specification. It is to be noted, however, that the appended

drawings illustrate preferred embodiments of the invention an d

therefore are . not to be considered limiting in their scope.

Figure I A shows the targeted (active) delivery of

particles to tumor blood vessels. Figure IB shows the interaction of the particles with laser or ultrasonic radiation.

Figure 2A shows a laser system for enhancement of drug

delivery in solid tumors resulted from hot cavitation and local heating

induced by interaction of laser radiation with particles. Figure 2 B

shows an ultrasound system for enhancement of drug delivery in solid

tumors resulted from cold cavitation and acoustic streaming induced

by interaction of ultrasonic radiation with particles.

Figure 3A shows interstitial irradiation of a deeply

located tumor with particles by laser radiation delivered through a n

optical fiber inserted in a needle. Figure 3B shows laser irradiation

of a tumor with particles in a hollow organ via an endoscope. Figure

3C shows irradiation of a tumor with particles in a hollow organ by a n

ultrasonic transducer inserted in the organ.

Figure 4 shows particle penetration in rat liver tissue.

Figure 4 A shows the penetration due to irradiation by nanosecond

Nd:YAG laser pulses. Figure 4B shows the penetration due to

irradiation by nanosecond Alexandrite laser pulses. Figure 4C shows

the penetration without laser irradiation (control).

Figure 5 shows fluorescence spectra of fluorescein

isothiocyanate-dextran (FITC-Dextran) penetrated into the liver tissue

due to irradiation by nanosecond Nd:YAG laser pulses (upper

spectrum), Alexandrite laser pulses (middle spectrum) and without laser irradiation (lower spectrum).

Figure 6A shows a gross picture of muscle tissue with

carbon (left) and graphite (right) particles penetrated in the

interstitium due to sonication for 10 minutes. Figure 6B shows

carbon particle penetration into the tissue due to 10-min. sonication.

Figure 6C shows a carbon particle penetration into the tissue after 3 -

min. sonication. Figure 6D shows carbon particle penetration in the

tissue without sonication.

Figure 7 shows a fluorescence spectra of FITC-Dextran

penetrated in the muscle tissue due to 10-min. sonication and without

sonication.

Figure 8 shows particle penetration due to 10-min .

sonication in liver (Figure 8A ), kidney (Figure 8B) and lung tissues

(Figure 8C) :

Figure 9 shows fluorescence spectra of FITC-Dextran.

Figure 9 A shows penetration in the liver tissue due to 10-min .

sonication and without sonication. Figure 9B shows penetration in

the liver, lung, and kidney tissue due to 10-min. sonication.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "electromagnetic radiation (wave)" refers to radiation (wave) with electrical and magnetic component which

includes (but not limited to) optical (ultraviolet, visible, and infrared

light), microwave, and radiofrequency radiation (wave).

As used herein, "ultrasound" or "ultrasonic radiation

(wave)" refers to mechanical ("acoustic" or in other terms of

"pressure") wave in a medium in the frequency range from 20 kHz to

about 1 GHz.

"Nanoparticles" refers generally to particles with a

diameter from 0.1 to several hundred of a nanometer. 1 nanometer =

10^"9 meter. "Microparticles" generally refers to particles with a

diameter from 0.1 to several hundred of micrometer. 1 micrometer =

10^"6 meter. As used herein, the term "nanoparticles" refers to

particles with a diameter from about 0.1 to about 7000 nm.

As used herein, "cavitation" refers to a physical

phenomenon in a liquid or a liquid-like medium (including tissue)

represented by formation of vapor (or gas) bubbles followed b y

growth, oscillation, and collapse of the bubbles.

As used herein, "acoustic streaming" refers to a physical

phenomenon in a liquid or a liquid-like medium represented by flows

of a part of the liquid medium upon irradiation by an ultrasonic wave.

As used herein, "minimal fluence" refers to lowest possible

fluence of laser radiation (measured in J/cm²) to induce drug delivery enhancement without damage to normal tissues.

As used herein, "microconvection" refers to micron- sized

displacement (flow) in liquid. Generally, convection refers to

movement (flow) of a part of a liquid medium.

As used herein, "antibody coating" refers to a coating of a

particle due to attachment of a number of antibody molecules to the

particle.

As used herein, "particle migration" refers to movement

(travel, displacement) of particles. It is cased by cavitation, streaming ,

or local heating effects.

The purpose of the invention is to disclose means for

enhancement of drug delivery through the physiological barriers of

solid tumors, such as (1) a tumor blood vessel wall; (2) the

interstitium; and (3) a cancer cell membrane. Such methods utilize

interaction of electromagnetic or ultrasonic radiation with

nanoparticles and microparticles selectively accumulated in tumor

blood vessels. The interaction alters the physiological properties of

the barriers of solid tumors due to: (1) cavitation induced b y

ultrasonic, pulsed laser, or microwave radiation near the particles; (2)

transient local heating of the particles by laser or microwave

radiation; and (3) acoustic streaming near the particles. The physical

forces result in rupture of tumor blood vessel wall and cancer cell membrane, and microconvection in the interstitium leading to

enhanced delivery of the macromolecular drugs into cancer cells.

Nanoparticles or microparticles having certain physical

properties and capable of inducing cavitation, local heating, or acoustic

streaming upon irradiation by electromagnetic or ultrasonic radiation

are intravenously injected and selectively accumulated in tumor

vasculature. Passive or active delivery of long-circulating particles

provides a selective accumulation of the particles in tumor

vasculature. The particles are coated with polyethylene glycol or

polyethylene oxide to provide long circulation in blood. Recently

discovered antibodies and short peptide sequences can be used for

active delivery of the particles to tumor blood vessels. Experiments

on animal models demonstrated the feasibility of active delivery of

long-circulating nanoparticles loaded with anti-cancer drugs an d

coated with antibodies that results in their selective accumulation in

tumors .

Cavitation (formation, growth, oscillations, and collapse of

microbubbles) is initiated by the nano- or microparticles w hen

ultrasonic or electromagnetic radiation is applied. A source of

radiation is placed on the surface of skin or inserted in a hollow organ

or in the interstitium so that the irradiation of the particles in the

tumor is provided. Cavitation is known as both "cold" cavitation, induced b y

tensile (negative) pressure of ultrasonic waves and "hot" cavitation,

induced by evaporation upon heating of an absorbing medium b y

electromagnetic radiation. Ultrasonic irradiation results in cavitation

near the particles which serves as cavitation nuclei significantly

reducing cavitation threshold. The material, structure, and dimensions

of the particles and ultrasound frequency are specially selected to

provide cavitation effects at minimal power of the ultrasonic wave.

Pulsed electromagnetic (laser or microwave) irradiation

results in evaporation of blood near the particles strongly absorbing

optical or microwave radiation. The material, structure, an d

dimensions of the particles as well as radiation wavelength and pulse

duration are specially selected to provide cavitation effects at minimal

fluence of laser or microwave radiation. This yields cavitation

confined within the tumor and with minimal mechanical or thermal

damage to normal tissues.

Perforation of tumor blood vessel walls is induced by the

growing, oscillating, and collapsing cavitation bubbles. As a result,

anti-cancer drugs circulating in blood penetrate through the tumor

blood vessel wall into the interstitium. The cavitation bubbles also

induce microconvection in the interstitium. Anti-cancer drugs migrate

in the interstitium due to microconvection. Perforation of cell membrane is induced by the cavitation bubbles, which allows th e

anti-cancer drugs to penetrate from the interstitium into the cancer

cells. Therefore, the death of cancer cells in the tumor is caused b y

chemical or biological effects of anti-cancer drugs; mechanical or

thermal damage to the cancer cells from cavitation bubbles; an d

mechanical or thermal damage as well as chemical and biological

damage to the tumor vasculature from cavitation bubbles and the

drugs .

In one embodiment of the present invention, there is

provided a method of enhancing anti-cancer drug delivery in a solid

tumor, comprising the steps of administrating at least one anti-cancer

drug to the tumor; injecting nanoparticles or microparticles to the

tumor intravenously; and irradiating the tumor with radiation.

Generally, the anti-cancer drug is selected from the group

consisting of a monoclonal antibody, a cytokine, an antisense

oligonucleotide, a gene-targeting vector and any other macromolecular

therapeutic agent. Generally, the tumor occurs in the organ selected

from the group consisting of breast, lung, brain, liver, skin, kidney, GI

organ, prostate, bladder, gynecological organ and any other hollow

organ .

Preferably, the nanoparticles or microparticles are long

circulating particles with or without antibody coating, wherein the antibody is directed against tumor vasculature. The nanoparticles or

microparticles can be metal particles, carbon particles, graphite

particles, polymer particles loaded with an absorbing dye, liquid

particles loaded with an absorbing dye or porous particles having gas-

filled pores. The nanoparticle has a diameter from about 0.1 nm to

about 7000 nm.

Preferably, the radiation is optical pulsed radiation

generated from a laser or non-laser source. Specifically, the optical

radiation is in the spectral range from 0.2 μm to 2 μm and delivered

through the skin surface or via optical fibers inserted in a needle or

endoscopes to the tumor.

Preferably, the radiation is ultrasonic radiation generated

from an ultrasonic transducer. Specifically, the ultrasonic radiation is

in the frequency range from 20 to 500 kHz and delivered through th e

skin surface to the tumor.

In another embodiment of the present invention, there is

provided a system for enhancement of anti-cancer drug delivery in a

solid tumor, comprising a source of radiation; an electronic system or

means for monitoring of the radiation; a system for delivery of th e

radiation to the tumor; nanoparticles or microparticles absorbing the

radiation; an injection system or means for administration of the anti-

cancer drug and the nanoparticles or microparticles in tumor blood. In still another embodiment of the present invention,

there is provided a method of treating a solid tumor without anti-

cancer drugs, comprising the steps of injecting nanoparticles or

microparticles to the tumor intravenously and irradiating the tumor

with radiation.

The following examples are given for the purpose of

illustrating various embodiments of the invention and are not me ant

to limit the present invention in any fashion.

EXAMPLE 1

Short Laser Pulses

It is known that severe local heating of strongly absorbing

particles in transparent optical materials produces vapor

microbubbles upon irradiation by short laser pulses, which results i n

mechanical and thermal damage to the materials. Local heating of a

strongly absorbing particle in a medium can be induced, if laser pulse

duration is shorter than the time of heat diffusion. The heat diffusion

2 time, τ, is estimated as τ ~ d /kχ , where d is the dimension of th e

particle, k is a coefficient which is dependent on the shape of the

particle, and χ is the thermal diffusivity of the medium. For example,

local temperature rise above 20,000°K can be achieved in laser glass containing a metal particle with the radius of 200 nm and absorption

coefficient of about 10 cm^" ! upon irradiation with nanosecond laser

2 pulses with the fluence of 20 J/cm .

Local heating of exogenous strongly absorbing

nanoparticles by short (nanosecond) and ultrashort (picosecond) laser

pulses results in explosive evaporation of blood in tumor vasculature

and formation of microbubbles.

EXAMPLE 2

Active and Passive Delivery

To avoid rapid clearance by the reticuloendothelial system

and obtain selective accumulation in tumors, long-circulating particles

coated with a surfactant (polyethylene glycol or polyethylene oxide)

are used. The long-circulating particles are attached to antibodies

directed against antigens in tumor vasculature, which results in

selective delivery of the particles to tumor blood vessel walls (active

delivery) (see Figure IA). Short peptide sequences for organ specific

targeting may be used for this purpose. The long-circulating particles

without antibodies also selectively accumulate in tumors because of

leakage through tumor vasculature (passive delivery). Figure IB

shows the interaction of the particles with pulsed laser or ultrasonic radiation. Cavitation induced by laser or ultrasonic radiation results in

perforation of tumor blood vessels and penetration of the particles

and anti-cancer drug into the interstitium due to microconvection.

The cavitation-induced microconvection provides delivery of the

particles and anti-cancer drug through the interstitium to cancer cell

membranes. Perforation of the cell membranes allows the drug

molecules to penetrate into the cancer cells.

EXAMPLE 3

Systems for Enhancing Drug Delivery in Solid Tumors

A laser system for the enhancement of drug delivery in

solid tumors was developed (see Figure 2A). The system utilizes

nanosecond Nd:YAG laser radiation with the wavelength of '1064 nm to

induce local heating of strongly absorbing particles. The s ystem

consists of: (1) Nd:YAG laser with electronic system for monitoring of

radiation parameters (pulse energy and duration, number of pulses ,

repetition rate, power); (2) a fiber-optic system or conventional optical

system for delivery of laser radiation through the skin to the tumor;

(3) nano- or microparticles strongly absorbing laser radiation an d

attached to antibodies directed against tumor vasculature; and (4) a n injection system for administration of the particles and anti-cancer

drugs in blood.

An ultrasound system for the drug delivery enhancement

utilizing ultrasonic waves capable of inducing cavitation in blood an d

tissues was also developed (see Figure 2B). The system consists of: ( 1 )

ultrasonic transducer (or transducer array) for producing ultrasonic

wave with a frequency in the range from 20 kHz to 10 MHz; (2) a n

electronic system for providing power supply for the ultrasonic

transducer and monitoring of radiation parameters (pressure

amplitude, frequency, pulse duration, number of pulses, repetition

rate, ultrasonic power); (3) a focusing system for directing an d

focusing ultrasonic radiation to tumors; (4) nano- or microparticles

decreasing threshold of cavitation upon irradiation by the ultrasonic

radiation and attached to antibodies directed against tumor

vasculature; and (5) an injection system for administration of th e

particles and anti-cancer drugs in blood.

EXAMPLE 4

Interstitial Irradiation of Tumors

Near infra-red radiation with sufficient fluence can penetrate only up to approximately 2 - 4 cm into tissues depending on

their optical properties. To overcome this problem, optical fibers

inserted into needles can be used for irradiation of deeply located

tumors (so-called interstitial irradiation of tumors). Figure 3 A

illustrates a laser system utilizing the interstitial irradiation of tumors .

An optical fiber inserted into a needle is introduced into a tumor an d

laser radiation is not attenuated by tissues between the tumor an d

skin surface. This provides laser irradiation of the tumor with th e

fluence sufficient to enhance drug delivery.

A laser system utilizing irradiation of tumors in the lung,

stomach, intestine, and other hollow organs through optical fibers or

endoscopes is shown in Figure 3B. Near infra-red, visible, or near-UV

laser radiation is used in this case because the tumors are irradiated

directly and without attenuation in the skin and normal tissues.

Figure 3C illustrates an ultrasound system utilizing

transducer incorporated in an endoscope for irradiation of tumors in

hollow organs. The tumor is directly irradiated by the transducer that

allows more efficient drug delivery. The systems shown in the Figures

3 A, 3B and 3C provide selective irradiation of solid tumors at lower

incident laser pulse energy and ultrasonic power that minimizes

damage to normal tissues. EXAMPLE 5

Laser Irradiation-Induced Particle Penetration in Rat Liver Tissue

Freshly excised ex vivo rat liver was used. Activated

carbon particles with the diameter of 1 μm were used as particles

strongly absorbing Nd:YAG laser radiation with the wavelength of

1064 nm. The particles were placed between two liver slabs with th e

dimensions of 10 x 10 mm. The thickness of each slab was 3 mm. The

slabs were irradiated for 10 minutes by the laser pulses with the

energy of 90 mJ, duration of 15 ns, repetition rate of 10 Hz. The laser

spot diameter was 8 mm yielding incident fluence of 0.18 J/cm². The

slabs were placed in 1% water solution of fluorescein isothiocyanate-

dextran (FITC-Dextran) to study penetration of macromolecules in the

liver tissue. Molecular weight of the FITC-dextran is 12,000. After

irradiation the slabs were rinsed in pure water to remove the particles

and dextran from the tissue surface. Laser-induced explosive

evaporation of water results in penetration and migration of the

particles and dextran in the interstitium (see Figure 4A). The black

spots represent clusters of the carbon particles. Magnification is x

350. Particle penetration is up to 160 μm .

Particle penetration in the rat liver tissue due to

irradiation by nanosecond Alexandrite laser pulses with the wavelength of 720 nm was also studied (Figure 4B). The liver slabs

were irradiated for 10 minutes by the laser pulses with the energy of

90 mJ, duration of 160 ns, repetition rate of 2 Hz. All other conditions

are the same as in the case of Nd:YAG irradiation. The laser spot

diameter was 4 mm yielding incident fluence of 0.72 J/cm².

Magnification is x 460. Particle penetration is up to 60 μm .

As in a sample without application of laser irradiation

(control), some particles are visible only on the tissue surface (Figure

4C). However, the particles can not penetrate into the interstitium.

Magnification is x 460.

Fluorescence spectra of FITC-Dextran penetrated in the

liver tissue was further studied (Figure 5). All conditions are the

same as described in the Figure 4. The wavelength for the

fluorescence excitation is 400 nm. Increased penetration of th e

dextran molecules is obtained upon Nd:YAG laser irradiation (4-fold in

comparison with the non-irradiated sample). Dextran penetration

upon Alexandrite laser irradiation is also noticeable (2-fold i n

comparison with the non-irradiated sample).

These data demonstrate that interaction of pulsed laser

radiation with strongly absorbing particles results in penetration of

the particles and macromolecules into the interstitium and migration

of the particles and macromolecules in the interstitium. EXAMPLE 6

Sonication-Induced Particle Penetration in Chicken Muscle Tissue

Particle penetration due to sonication was conducted in

chicken muscle tissue (Figures 6A, 6B and 6C). Figure 6A shows a

gross picture of chicken muscle tissue with carbon (left) and graphite

(right) particles penetrated in the interstitium due to sonication for 1 0

minutes. The activated carbon and graphite particles with the

diameter of 1-2 μm were used as cavitation nuclei. The activated

carbon particles have numerous pores filled with air that substantially

decrease the cavitation threshold and enhances cavitation. Ultrasonic

radiation with the frequency of 50 kHz and incident intensity of 250

mW/cm² was employed for the experiments. Penetration of the

activated carbon particles is more pronounced. No cavitation w as

visible on the tissue surface where the particles were not applied.

Also shown is that carbon particle penetrates into the

tissue up to 180 μm due to 10 minute sonication (Figure 6B,

Magnification: x 230) and up to 60 μm due to 3 minute sonication

(Figure 6C, Magnification: x 300). Particles are visible only on the

tissue surface without penetrating into the interstitium when no

sonication was applied (Figure 6D, Magnification: x 340).

Fluorescence spectra of FITC-Dextran penetrated in th e muscle tissue due to 10-min. sonication and without sonication w as

also conducted (Figure 7). All conditions are the same as in the Figure

6. The data demonstrate more than 6-fold increase of the penetration

of the macromolecules into the tissue which is caused by ultrasound-

induced cavitation. The cavitation results in microconvection in the

tissue leading to enhanced delivery of macromolecules in th e

interstitium.

EXAMPLE 7

Sonication-Induced Particle Penetration in Rat Tissues

Particle penetration due to 10 minute sonication w a s

studied in freshly excised rat tissues ex vivo (Figures 8A, 8B and 8C,

Magnification: x 460). The penetration is up to 160 μm in the liver, u p

to 30 μm in the kidney, and up to 150 μm in the lung tissue.

Fluorescence spectra of FITC-Dextran penetrated in the r a t

liver tissue due to 10-min. sonication and without sonication was

further conducted (Figure 9A). The intensity of fluorescence from the

sonicated tissue is greater 6.7 times than the one from the n on-

sonicated. Figure 9B shows fluorescence spectra from liver, lung, an d

kidney tissue after 10-min. sonication. The intake of the dextran

molecules is almost the s ame for all the tissues. The ultrasound d ata demonstrate that the interaction of the particles with ultrasonic

radiation induces cavitation in tissues and the cavitation results in

penetration of particles and macromolecules into the interstitium.

Discussion

The present invention utilizes the interaction of laser or

ultrasonic radiation with exogenous intravenously injected particles

for the purpose of enhancing drug delivery in solid tumors. The

interaction enhances penetration of anti-cancer drugs, especially

macromolecular therapeutic agents, from blood into cancer cells

preferably due to cavitation accompanied by disruption of tumor

blood vessel walls and cancer cell membranes as well a s

microconvection in the interstitium.

Local heating of exogenous strongly absorbing

nanoparticles by short (nanosecond) and ultrashort (picosecond) laser

pulses results in explosive evaporation of blood in tumor vasculature

and formation of microbubbles. Optical radiation in the near infra-red

and visible spectral range (so-called "therapeutic window": λ = 600 -

1300 nm) has low attenuation in tissues. Therefore, it can induce local

heating of the strongly absorbing particles in deeply located tumors

without damage to irradiated tissue surface. For example, absorption

and scattering coefficients of breast tissue equal to 0.05-0.08 cm an d 5.0-9.0 cm , respectively, in the near infra-red spectral range. Using

- 1 -2 2 these values, one can estimate that the fluence of 10 - 10 J/cm can

be achieved at the depth of 2-3 cm, if breast tissue is irradiated b y

the nanosecond Nd:YAG laser with a pulse energy of 1 J and a beam

diameter of 1 cm. Since temperature rise is proportional to fluence,

local transient temperature of about 50- 140 C can be obtained near

the particles.

Overheating of water above 100°C near absorbing particles

induces local explosive microevaporation resulting in pressure rise,

bubble formation, and outward momentum. Collapse of the

microbubbles induces strong inward momentum and pre s sure

transients. Both the explosive evaporation and the following bubble

collapse perforate tumor vasculature and therefore increase vascular

permeability, which allows therapeutic agents to penetrate from blood

into the interstitium, migrate in the interstitium, and penetrate into

the cancer cells. Moderate heating without evaporation can induce

local thermal damage or local temperature rise without damage which

may also increase vascular permeability. Pulsed laser heating of

absorbing volumes generates thermoelastic pressure transients in

tissues which may contribute to damaging tumor vasculature or

increasing vascular permeability. Possible photochemical an d

thermochemical reactions and ionization induced by the absorbing nanoparticles may also enhance drug delivery in solid tumors.

Low absorption of optical radiation by tissues and strong

absorption by particles enhances drug delivery in tumors without

damage to normal tissues. Laser ablation of the irradiated tissue

2 surface is not induced, because the incident fluence of about 1 J/cm is

substantially lower than the tissue ablation threshold of 40-45 J/cm

at this wavelength. The particles should have high absorption

4 6 - 1 coefficient ( 10 - 10 cm ), low emissivity, short lifetime of excited

states, capability to bind to antibodies, low acute and chronic toxicity.

Potential candidates are carbon, graphite, or metal (gold, silver,

platinum) particles. Solid particles injected intravenously have low

acute and chronic toxicity. Ideally, particles used for the drug

delivery enhancement should be biodegradable. Insoluble liquid (for

instance, liposomes) or biodegradable polymer particles can be stained

with a strongly absorbing dye and used for the drug delivery

enhancement instead of carbon or metal particles.

The ultrasonic "cold" cavitation enhances drug delivery

from blood into cancer cells as in the case of laser-induced "hot"

cavitation. The cavitation is induced by tensile (negative) pressure of

the ultrasonic, wave. The ultrasonic wave propagates through the skin

and normal tissues with insignificant attenuation and induces

cavitation upon interaction with the particles. It is known th at cavitation threshold is substantially lowered by cavitation nuclei. The

particles selectively delivered in tumor blood vessels and the

interstitium are used as the nuclei decreasing cavitation threshold.

Porous particles (such as activated carbon particles) with gas-filled

pores can substantially lower cavitation threshold because they

already have initial ("seed") bubbles.

Combination of laser-induced "seed" cavitation and the

following bubble growth and collapse caused by ultrasonic waves can

be applied for more efficient drug delivery in solid tumors. In this

case, laser radiation initiates bubble formation producing cavitation

nuclei while ultrasonic radiation increases dimensions of the bubbles,

induces their oscillations and collapse resulting in microconvection.

Ultrasonic waves with moderate amplitude induce acoustic

streaming (microconvection) near particles without cavitation, if their

acoustic impedance is different from that of surrounding medium.

The acoustic streaming may also enhance delivery of therapeutic

agents from blood into tumor cells.

Pulsed microwave and radio-frequency radiation can

induce local heating of absorbing particles (e.g., metal), because of its

deep penetration into tissues and strong absorption in metals.

However, the difference in absorption between metals and tissues is

substantially less than in the case of visible or near infra-red radiation. In addition, generation and delivery of powerful short

microwave or radio-frequency pulses to tumors are more complicated

than the generation and delivery of short optical pulses.

Thermal and mechanical damage to endothelial cells of

tumor vasculature induced by laser and ultrasonic radiation can b e

used for cancer treatment without drugs. Such damage results in the

lack of blood supply to the tumor with the following avalanche d eath

of cancer cells. Particle migration in the interstitium caused b y

interaction with laser or ultrasonic radiation induces thermal an d

mechanical damage to cancer cells that may result in death of cancer

cells. Such an approach eliminates side effects of chemotherapy.

The following publications were referred to herein.

US Patent No. 5,474,765 Thorpe

US Patent No. 5,487,390 Cohen et. al.

US Patent No. 5,543,158 Gref et. al.

US Patent No. 5,651 ,986 Brem et. al.

US Patent No. 5,565,215 Gref et. al.

US Patent No. 5,578,325 Domb et. al.

US Patent No. 5,614,502 Flotte et. al.

US Patent No. 5,658,892 Flotte et. al.

US Patent No. 5,660,827 Thorpe et. al.

US Patent No. 5,718,921 Mathiowitz et. al. US Patent No. 5,713,845 Tankovich

US Patent No. 5,762,918 Thorpe

US Patent No. 4,971 ,991 Imemura et. al.

US Patent No. 5,403,590 Forse

US Patent No. 5,380,411 Schlief

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Gref R., et al., Science v. 263, pp. 1600-1603, 1994.

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Thorpe, et al., Breast Cane. Res. and Treatm. 36 (2), 237-251 , 1995.

R. Pasqualini, et al., Nature v. 380, pp. 364-366, 1996.

W. Arap, et al., Science v. 279, pp. 377-380, 1998.

Takahashi T., Crit. Rev. Ther. Drug Carr. Syst. 2(3), pp. 245-274, 1986.

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Johnson M. E., et al, J. Pharm. Sci. v. 85 (7), pp. 670-679, 1996.

Folkman J., Sci. Am. v. 275, pp. 150-154, 1996.

Esenaliev R. O.; et al., Appl. Phys. B v. 59, pp. 73-81 , 1994.

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Any patents or publications mentioned in this specification

are indicative of the levels of those skilled in the art to which the

invention pertains. These patents and publications are herein

incorporated by reference to the same extent as if each individual

publication was specifically and individually indicated to b e

incorporated by reference.

One skilled in the art will readily appreciate that the

present invention is well adapted to carry out the objects and obtain

the ends and advantages mentioned, as well as those inherent therein.

The present examples along with the methods, procedures, treatments ,

molecules, and specific compounds described herein are presently

representative of preferred embodiments, are exemplary, and are not

intended as limitations on the scope of the invention. Changes therein

and other uses will occur to those skilled in the art which are

encompassed within the spirit of the invention as defined by the scope

of the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing anti-cancer drug delivery in

a solid tumor, comprising the steps of:

administering at least one anti-cancer drug to said tumor;

injecting nanoparticles or microparticles to said tumor

intravenously; and

irradiating said tumor with radiation.

2. The method of claim 1 , wherein said anti-cancer

drug is selected from the group consisting of a monoclonal antibody, a

cytokine, an antisense oligonucleotide, and a gene-targeting vector.

3. The method of claim 1, wherein said tumor is in an

organ selected from the group consisting of breast, lung, brain, liver,

skin, kidney, GI organ, prostate, bladder and gynecological organ.

4. The method of claim 1 , wherein said nanoparticles or

microparticles are long-circulating particles with or without antibody coating.

5. The method of claim 4, wherein said antibody is

directed against tumor vasculature.

6. The method of claim 1 , wherein said nanoparticle h as

a diameter from about 0.1 nm to about 7000 nm.

7. The method of claim 1 , wherein said nanoparticle or

microparticle is selected from the group consisting of a metal particle,

a carbon particle, a graphite particle, a polymer particle, a liquid

particle and a porous particle.

8. The method of claim 1 , wherein said radiation is

optical pulsed radiation generated from a laser or non-laser source.

9. The method of claim 8, wherein said optical radiation

is delivered through skin surface or via optical fibers or endoscopes to said tumor

10. The method of claim 8, wherein said optical radiation

is in the spectral range from 0.2 ╬╝m to 2 ╬╝m .

1 1 . The method of claim 1 , wherein said radiation is

utrasonic radiation generated from an ultrasonic transducer.

12. The method of claim 11 , wherein said ultrasonic

radiation is in the frequency range from 20 to 500 kHz.

13. The method of claim 11 , wherein said ultrasonic

radiation is delivered through skin surface to said tumor.

14. An anti-cancer drug delivery system, comprising:

a source of radiation;

an electronic system for monitoring of said radiation; a means for delivery of said radiation to said tumor;

nanoparticles or microparticles absorbing said radiation;

an injection means for administration of said anti-cancer

drug and said nanoparticles or microparticles in blood.

15. A method of treating a solid tumor, comprising the

steps of:

injecting nano- or microparticles to said tumor

intravenously; and

irradiating said tumor with radiation.