INTRODUCTION:

Molecular imaging which was emerged in the early 21^st century has shown an immense growth and it is a technique which visualises the cellular functions along with molecular processes occurring in living organisms. From molecular imaging [2], ultrasound based molecular imaging has come into light, which has given a hope for the clinical experts. This ultrasound molecular imaging is a real-time non-invasive, cost-effective and it is been widely used in the recent times. For this technique ultrasound contrast agents like microbubbles and nanobubbles are been used. Micro and nanobubbles has been a promising non-viral tool for ultrasound mediated drug delivery. These have attracted and pave way for the research of many drugs. As microbubbles due to their limited particle size, they couldn’t pass through the blood vessels and remain as blood pool agents [5]. This led to consider the nano scale system such as nanobubbles that can extravascate from blood vessels into the surrounding tissues and thus enhancing the localization and drug delivery efficiency. Micro and nanobubbles along with the ultrasound mediation have been widely used especially in the cancer therapy, in order to achieve greater therapeutic efficiency with lesser adverse effects. This review summarizes detail knowledge on Micro and nanobubbles structure, formulation design and various preparation methods along with the research details regarding the different drugs that are loaded in micro or nanobubbles to attain better results in the drug delivery of various diseases.

Advantages of micro and nanobubbles:

1. Non-invasiveness, low toxic, targeted and local application, cost effective technique [19].

2. These are useful for theranostic delivery.

3. Act as a therapeutic tool for antibodies delivery, oxygen delivery, nucleic acid delivery [7].

4. This technique is been widely used for delivery of anticancer drugs [7].

Disadvantages of micro and nanobubbles:

1. It is a multistep process.

2. Pore size may be a limiting factor which hinders the transport of the therapeutic agents through the cell membrane.

STRUCTURE OF MICRO/NANOBUBBLE

The structure of micro/ nanobubble consists of mainly core and shell, where the shell plays a major role in bubble formation.

Core -	Air, oxygen, sulphur hexafluoride, perfluorocarbons
Shell -	Surfactant/co- surfactant, lipid, polymer, protein, polyelectrolyte multilayer.

Shell: The biocompatibility, stability and the release of core gas of the micro/nanobubbles depends mainly on the type of shell and the core gas being used. The shell is of three types [6], lipid shell (3 nm), protein shell (15-20 nm) and polymer shell (100-200 nm). Shell properties are as follows:

· Shell forms protective layer around gas in order to provide stability.

· It provides protection from endogenous scavengers.

· It reduces the rate of diffusion of the core gas into the surroundings.

· Shell composition plays an important role in loading of drugs.

· The composition of the shell determines the elasticity, gas exchange, resistance against applied ultrasonic pressure, stiffness, half-life and also mainly ease in excretion of micro/nanobubbles from body.

The composition of shell plays a major role because if the shell is soft it would break easily, and if it is hard it would not be able to oscillate in the ultrasonic fields. So, appropriate shell materials have to be taken based on the applications of micro/nanobubbles. Depending upon the shell composition the in vivo half-life of micro/nanobubbles varies from few seconds to several hours.

Properties of various shell types:

Lipid shell: These are flexible shells and are 3 nm in thickness, formed by lipids. Under acoustic pressure, they show improved resonance and allow diffusion of gases from shell [6] is highly cohesive providing solid like character. Mostly used lipids are phospholipids, which are amphiphilic in nature having a hydrophilic head and an hydrophobic tail. The structure of lipid shell is shown in Fig. 1.

Examples of various combinations of base phospholipids: 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) these are used in combination with various emulsifiers and surfactants. Another compound which is highly hydrophilic, low toxicity, improves stability and avoids coalescence is PEG (polyethylene glycol). This PEG incorporated into lipid reduces the immunogenic responses, prolongs circulation, decrease plasma clearance and increases half-life. The structure of lipid shell is shown in Fig. 1.

Fig. 1: Structure of lipid shell [18].

Protein shells: These are prepared by heating the protein solution to the denatured point and then followed by emulsification. The denatured protein[6] forms thin mono layer shell across the desired gas. They contain bubbles with a diameter of 1-15 µm and shell thickness of 15 nm. The structure of protein shell is shown in Fig. 2.

Fig. 2: Structure of protein shell [18].

Advantages:

1. These are favourable for biocompatibility, biodegradability, amphipathic nature, stability and longer half-life.

2. Protein-shelled oxygen microbubbles have been used for oxygen delivery to oxygen depleted saline solution.

Examples: For ultrasound contrast agent albumin shelled micro/nanobubbles is used. Commercial products like ALBUNEX are approved by the Food and Drug administration and used for commercial applications.

Polymer shells: These are considering being more resistant during the application of ultrasound fields, and are thicker than protein and lipid shells, they show dampened response [6] in the acoustic fields. The range of these polymeric shells are 150-200 nm, therefore it enables higher drug loading capacity of hydrophilic and hydrophobic drugs. The structure of polymer shell is shown in Fig. 3.

Advantages: They exhibit good stability, biocompatibility, biodegradability and purity.

Examples: The following are used to synsthesie polymeric shell micro/nanobubbles: Chitosan, PLA (polylactic acid), PVA (poly vinyl alcohol), PGA (poly glycolicacid), PLGA (poly-lactic-co-glycolicacid).

Fig. 3: Structure of polymer shell [18].

FORMULATION DESIGN OF MICRO/ NANOBUBBLES

The formulation design is a challenging task because number of factors has to be taken into consideration. Role played by interfacial tension and laplace pressure is to be considered firstly and then structure of dispersed and continuous phase. The surface tension at the interface of binary mixtures is defined as the molecular interaction [7]between the internal gas core and outer liquid medium, given in Eq. 1.

ΔP=P_inside-P_outside=2σ/r

Eq. 1

Where: P_inside=Pressure inside bubble, P_outside= Pressure outside bubble, σ= interfacial tension and r= Bubble radius. The pressure difference between the inside and outside of a bubble or a droplet is called as Laplace pressure. The laplace pressure is inversely proportional to the bubble size, i.e smaller bubble have higher pressure values. To make bubble stable, it includes various approaches [7]:

· Reduction of laplace pressure difference.

· Limitation of gas diffusion.

· Presence of surfactants at interface.

· Control of interfacial structure.

By adding surfactants to the formulation will lead to formation of molecular monolayer at the interface which can decrease interfacial tension and limit the pressure difference between inside and outside of bubble.

There is another strategy for designing nanobubble, it involves usage of inert gas of very poor aqueous solubility for composition of core such as sulphur hexafluoride, perfluorocarbons, so that the dissolution rate of internal gas of the nanobubbles is reduced. Hence in this way, the solubility factor of the gas helps to maintain stability of the bubbles in the bloodstream.

MECHANISM OF MICRO AND NANOBUBBLE

The mechanism[8]involved in micro and nanobubbles are in following steps:

· Application of ultrasound

· Sonoporation

· Delivery of therapeutic agents into the targeted cells.

Application of ultrasound:

The ultrasound radiation can be given either continuous or pulsative. It is categorised into three [8]:

· High frequency: Ranges from 3-10 MHz- diagnostic ultrasound in clinical imaging.

· Medium frequency: Ranges from 0.7-3.0 MHz- therapeutic ultrasound in physical therapy.

· Low frequency: Ranges from 18-100 KHz - power ultrasound for cataract emulsification, liposuction and cancer therapy.

Microbubbles respond to ultrasound and exhibit robust dynamic behaviour [9], which is due to the high compressibility of the gas core. Microbubbles that are been used as ultrasound contrast agents consists of water insoluble gas core which is encapsulated by a thin layer of either lipid or polymer or protein, having thickness of about 10-100 nm. They are commercially available microbubbles where the encapsulated layer is made of lipid (Definity^TM, Sonovu^TM, Micromarker^TM) [9], or protein (denatured human albumin in Optison^TM). The main purpose of encapsulating layer is that it stabilises the bubble and prolongs lifetime by limiting gas diffusion and also decrease in surface tension and also highly responsive to ultrasound.

Sonoporation: It is the process of formation of small pores in the cell membrane by the application of ultrasound [9]. The effect of ultrasound has thermal and non-thermal effects [9], i.e. it includes cavitation, micro streaming, acoustic radiation forces and it is schematically represented in Fig. 4.

Cavitation - It refers to the formation of gaseous cavities in a medium by the application of ultrasound. It is responsible for growth and collapsibility of the bubbles. By application of the ultrasound, it leads to the formation of cavitation bubbles [9], and these will undergo contraction and expansion which leads to pushing and pulling on the cell and finally leads to opening of the pore. Inertial cavitation also called as transient cavitation is a process of formation, oscillation and collapsing of gaseous cavities driven by acoustic field. This transient cavitation occurs when the acoustic pressure is above a certain point of threshold and leads to volume expansion which is followed by collapsing during compression. The volume changes of the bubble will no longer follow the incoming ultrasound but it is dominated by inertia of surrounding liquid. This transient cavitation will also help to release the drugs and also micelle encapsulated drugs are easily delivered by this process.

Micro streaming - Rapid expansion and contraction will also generate fluid flow at the bubble which is in the form of micro streaming [9]. In some cases this micro streaming results in shear stress which will be exerted on adjacent cells leading to sonoporation, bubbles undergoing micro streaming play an important role in in vitro low frequency [11] sonophoresis.

Acoustic radiation forces - In addition to cavitation, acoustic radiation forces also affect bubbles and play an important role in sonoporation. During ultrasound molecular imaging procedure [10], these acoustic forces pushes the targeted bubbles toward the vessel wall and promotes binding and retention of the microbubbles at the diseased area. To ensure efficient translation of the bubble without causing destruction of bubble, the cavitation should be minimised by reducing the acoustic pressure as shown in Fig. 4.

Fig.4: Schematic representation of mechanism involved in sonoporation [9]. A-Induction of pushing by stable cavitation, B- Induction of pulling by stable cavitation, C- Induction of Jetting by inertial cavitation, D- Induction of microstreaming by stable cavitation, E- Induction of translation by primary acoustic radiation forces.

After the delivery of the therapeutic agent, the resealing process will also happen, in this resealing process calcium ions (Ca+) play an important role in cell repair.

PREPARATION TECHNIQUES FOR MICRO/NANO BUBBLES

The preparation techniques for micro/ nanobubbles are shown in Fig. 5.

Fig. 5: Represents preparation techniques for micro and nanobubbles.

MICROBUBBLE PREPARATION TECHNIQUES

1. High shear emulsification: In this method, emulsification of the polymer in an aqueous suspension occurs provided by high shear stirring [7]. The schematic representation of high shear emulsification process is shown in Fig. 6.

Fig. 6: Schematic representation of high shear emulsification process.

3. Sonication: It involves dispersing of gas or liquid in a suspension containing a suitable coating material, under high intensity ultrasound [7]. This leads to emulsification of the gas or liquid to form suspension containing microbubbles.

4. Ink-Jet printing: This method is mainly used to improve microbubble uniformity [12]. A stainless steel nozzle is supplied with fluid which is to be printed from a chamber in which a pirezoelectric crystal is embedded, by varying the voltage across the crystal, there is generation of pressure pulses in the liquid, with each pulse a droplet is forced out of the nozzle and more liquid is drawn into the chamber. These droplets can be collected in air or the nozzle may be submerged in a liquid filled container. The size of the droplet can be varied by changing the frequency or length of the pressure pulses rather than changing nozzle is advantage of this technique.

5. Coaxial electro hydrodynamic atomisation: In this a coaxial jet of two fluids are formed and are atomised to form uniform droplets [13]. And the fluids should be immiscible then only it is possible to encapsulate one fluid inside another, shown in Fig. 7.

Fig.7: Schematic representation of set up of co-axial electro hydrodynamic microbubbling.

It consists of two needles, the inner needle is supplied with gas and the outer needle is provided with suspension of desired coating material. Electric potential difference of several kilovolts is applied between the needles and an earthed ring electrode positioned at a short distance below their tips. The coaxial nozzle contains an inner needle with 150 µm inner diameter (ID) 300 µm outer diameter (OD). The outer needle dimensions are 685 µm (ID) and 1100 µm (OD). Both the needles used are stainless steel and are connected to the same applied voltage relative to an earthed ring electrode placed 12 mm below the outer needle. Outer diameter of ring electrode is 200 mm and inner diameter is 15 mm. Inner needle is placed approximately 2 mm above the tip of the outer needle. The glycerol and the air flow rates are controlled by high precision syringe pumps using plastic syringes. The syringes which are used are of 10 mL volume capacity for air and 5 mL for glycerol. The air and the liquid flow exiting the needles were monitored on screen using a LEICA S6D JVC colour video camera which is attached to a zoom lens and a data DVD video recorder. The bubbles are collected just below the ring electrode in a container of glycerol. Conditions: preparation should be carried out at atmospheric pressure and ambient temperature of 25 °C. This is the first technique to report that suspensions of microbubbles smaller than 10 µm, with a narrow size distribution.

6. Microfluidic device: It is well established method for the preparation of monodisperse liquid droplets and more recently have been used to prepare microbubble suspensions. There are two main types of microfluidic device [12]which have been used for bubble preparation:

I.Flow focusing units- lithography device.

II.Mechanically assembled units- T-junction device

Device consists of an orifice where column of gas impinges on a liquid flow and focused into a jet. At a certain distance from the orifice, the gas liquid interface becomes unstable and the bubbles are formed. Advantages of this technique are it provides smaller and larger bubble diameters and a high yield can be produced. It produces bubbles in a single step and can also produce multi-layer coating bubbles.

NANOBUBBLE PREPARATION TECHNIQUES

1. Sonication: Preparation of nanobubbles [14] by sonication process was shown in Fig. 8.

Fig. 8: Schematic representation of sonication process.

2. Centrifugation: This process involves ultrasonication [7]of a mixture of span 60 and polyoxyethylene 40 stearate (PEG 40S), which is followed by differential centrifugation to obtain a nanosized bubble having a unimodel size distribution.

3. Mechanical Agitation: Preparation of nano bubbles by mechanical agitation process [7] was shown in Fig. 9.

Fig. 9: Schematic representation of mechanical agitation process.

5. Manipulation of preformed microbubbles: This includes the preparation of microbubbles and these microbubbles [7] further undergoes filtration, floatation, centrifugation and condensation methods leading to the formation of nanobubbles.

6. Formulation ab initio of nanoscaled systems: This method is commonly used for the preparation of polymer coated micro-nanobubbles [7] which includes emulsification of the polymer in the aqueous suspension by high shear stirring, shown in Fig. 10.

Fig. 10: Schematic representation of formulation ab initio of nanoscaled systems.

CHARACTERIZATION OF MICROBUBBLES/ NANOBUBBLES

1. Concentration: The concentration of the bubble present per ml is counted by using Coulter counter machine [15].

2. Diameter and size distribution: The average diameter and size distribution of the bubbles can be determined by scanning electron microscopy, laser light scattering and transmission electron microscopy [15]. For example, the TEM images of PVA microbubbles and nanobubbles were shown in Fig. 11.

Fig. 11: Transmission electron microscope image of a) PVA microbubble [16]and b) Nanobubble [17].

4. Shell thickness: The thickness of the shell is determined by the coating of the shell with fluorescent dye like red nile and this is determined by fluorescent microscopy[15] against dark background.

5. Ultrasound reflectance measurement: It consists of transducer, bubble contained in a vessel consisting of metallic reflector and cellophane membrane [15], this vessel is in turn kept in another vessel containing water and the reflected signals are evaluated for the ultrasound reflecting capacity of bubble.

6. Air or Gas content by densitometry: The content of air encapsulated within the bubbles in suspension sample is measured by oscillation U-tube densitometry [15].

APPLICATIONS

1. Cardiac diseases treatment by micro and nanobubble: Ultrasound mediated microbubble suspension used as contrast agents has enabled better delineation of heart chambers and also visualizes myocardial microvasculature [20].

2. Gene delivery: Microbubble mediated targeted gene therapy combined with low frequency ultrasound is safe and efficient gene transfection technology. Wang LY et al., reported that in this therapy the microbubble mediated low frequency ultrasound enhanced the transfection [4] and expression of naked plasma DNA in cancer cells, and even improved the targeting of gene therapy with decrease in systemic side effects. Microbubbles are easily bound to genes by mixing them with gene vectors, And by wrapping/binding with microbubble shells, after the preparation they are injected into the body under the guidance of ultrasound. Due to ultrasound the microbubble gets destroyed and genes are released and bounded to the surface. Process of gene delivery was shown in Fig. 12.

Fig. 12: Different stages of DNA delivery using microbubbles and ultrasound [18].

4. Mediating targeted tumor drug therapy: The most important method to treat the tumor is chemotherapy. Moon et al., Ren et al., reported that when microbubbles carrying the drugs combined with ultrasound has shown an increase in concentration of chemotherapeutic drugs in tumor cells and also shown chemotherapeutic sensitization effect. Microbubbles which are containing the chemotherapeutic drugs are injected intravenously, and are irradiated with ultrasound. The microbubbles are targeted to the tissue and are ruptured leading to release of drug at targeted tissue [4], the low frequency ultrasound creates temporary gap in the cell membrane, leading to increase in permeability. The drug molecule enters the tumor cells, and improves the treatment efficiency.

5. Anti-tumor role: Proliferation and cloning of tumor cells are inhibited by microbubbles combined with low frequency ultrasound. The cells are inhibited by mechanical and cavitation effects. This inhibition leads to tumor cell necrosis, apoptosis Wang LY et al., reported that combination of microbubble with low frequency ultrasound directly suppressed prostate cancer cells evolution, and also promoted activation of anti-tumor immunocytes in VEGF-inhibited microenvironment.

6. Enhancement of drug delivery to Blood-brain barrier: the most challenging central nervous system disease is malignant glioma[4], which is associated with high rates of mortality. In the treatment process there is a temporary opening of the blood brain barrier and the therapeutic agents are delivered into the brain. Hao-Li Liu, et al., reported that the Microbubble induced focused ultrasound (FUS) blood brain barrier opening MB-FUS BBB opening serves as a promising method for non-invasively and locally enhancing the targeted delivery of therapeutic agents into CNS tumor regions, providing the potential to improve the treatment efficacy of chemotherapy.

7. Mediating sonothrombolysis: Acoustically meadiated disruption of blood clots is called as sonothrombolysis [3]. Normally thrombolytic agents are administered to dissolve the clots degrade them, but administration of microbubbles with ultrasound has increased the dissolution rate of clots, this process is studied for stroke, myocardial-infraction, venous thrombosis.

8. Kidney stones: Process of treatment of kidney stones by using micro and nano bubbles was shown in Fig. 13.

Fig. 13: Schematic representation of process of treatment of kidney stones by micro or nano bubbles: where A- Presence of stone in kidney, B- Blockage of stone in the ureter, C- Injecting of micro or nanobubble, D-Adheres to the stone, E- Application of ultrasound from outside, F- Vibration of the bubbles, G- Breaks the stone into tiny particles, H- Passes through ureter, I- Passes into the bladder and J- Fine particles in the bladder ready to excrete.

Table 1: List of research work carried out on micro and nanobubbles with various drugs incorporated into different research works [21-29].

S. No.	Drug	Category	Formulation	Disease condition	Results
1.	Doxorubicin (DOX)	Anticancer	Nanobubble	Breast cancer	Recent studies reported that water soluble chitosan oligomers can activate macrophages in presence of IFN-γ to kill cancer cells, so chitosan has direct and indirect anti tumor effects. DOX loaded biological chitosan nanobubbles are prepared and when used with ultrasound they could directly transport DOX into breast cancer cells [21].
2.	Apatinib	Tyrosine Kinase inhibitor anti-antiangiogenic agent	Nanobubbles	Gastric cancer	Apatinib is poorly water soluble, so loading into nanobubble shell increases its solubility and it is encapsulated to prevent killing of normal healthy cells. It actively targeted to HCC cells via the GPC-3 receptor, by carrying anti-GPC3antibody, drug is delievered to the cancer cells and thus enhancing the treatment efficiency [22].
3.	5-fluorouracil	Anticancer	Nanobubbles	Hepatocellular carcinoma	5-fluorouracil loaded nanobubbles combined with low frequency ultrasound imoroved targeted drug delivery and also effectively inhibited the growth of transplanted tumor [23].
4.	Doxorubicin	Anticancer	Nanobubbles	Human anaplastic thyroid cancer	Xenograft mouse model, which shown that there is greater acculumation of drug in tumor with consequent reduction of tumor volume and weight, causes extension of tumor doubling time with decreased cell proliferation [24].
5.	Docetaxel	Antineoplastic	Microbubble	Solid tumor	The toxicity and this DOC is not rapidly cleared from circulation after IV administration, so all can be solved by DOC loaded lipid micro bubble. DOC loaded lipid microbubble along with ultrasound targeted microbubble destruction lead to decrease of tumor cell proliferation and induce apoptosis which inhibits the growth of solid tumor and it is observed in mice [25].
6.	Doxorubicin loaded poly (Lactic-co-glycolic acid)	Anticancer	Nanobubble	Delivery of drug to HeLa cells	Doxorubicin is an anthracycline antitoxin and is effective chemotherapeutic anticancer drug, and treats various hematopoietictumors and solid tumors [26].
7.	Paclitaxel	Anticancer	Microbubble	Intraperitoneal ovarian cancer xenografts	Different treatment groups like the targeted and non-targeted ligand microbubbles have shown longer survival and significant therapeutic benefit with the mediation of the ultrasound. The destruction of the drug loaded microbubbles led to significant cytotoxicity and apoptosis effect on ovarian cancer cells A2780/DDP and without any adverse side effects in the animal [27].
8.	Doxorubicin (DOX)	Anticancer	Microbubble	Glioblastoma and breast cancer	It evaluated the applicability and effectiveness of administration of DOX combined with microbubble assisted ultrasound in glioblastoma and breast cancer cells [28].
9.	Doxorubicin (DOX)	Anticancer	Microbubble	Delivery of drug to cancerous cells	Involves two mechanisms where at first exposure of DOX-liposome loaded microbubble to ultrasound which leads to the release of free DOX and this free DOX is more cytotoxic than DOX-liposomes. Secondly the cellular entry of released DOX occurs which is facilitated due to sonoporation of cell membranes [29].

CONCLUSION:

Micro and nanobubbles combines with US have to be subjected to research for their potential use as therapeutic tool for clinical treatment. As they have many advantages and their structure also shows incorporation of high amount of potent active molecules and proves to show highly site specific release with wide range of therapeutic efficiency of drug delivery. Another added advantage of US targeted strategy is possibility of visualising the delivery of compound by real time ultrasound imaging. Finally we conclude that research should be processed on US mediated micro and nano bubble delivery and should observe the effect of this strategy on cells and tissues, interactions with plasma proteins, biodistribution and also toxicology aspects.

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Received on 20.04.2021 Accepted on 08.05.2021

International J. Technology. 2021; 11(1):6-18.

DOI: 10.52711/2231-3915.2021.00002