Nanoparticles in drug targetting Shyamkumar Immadi *

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Journal of Pharmacy Research Vol.5 Issue 2.February 2012

Review Article

ISSN: 0974-6943

Available online through

_{http://jprsolutions.info}

*Corresponding author. Shyamkumar Immadi

Bonn-Aachen International Center for Information Technology B-IT, DahlmannstraBe2, D-53113 Bonn, Germany.

1. Introduction

Designing drug delivery systems is challenging in terms of targeting drugs to specific sites. Certain chemicals or therapeutics agents that show success in cell culture fail to produce same effects in the human body because of the limitation in targeting to the designated area. As a result, high concentrations are given to patients, resulting into more side effects. This case is similar to biotechnological drugs such as recombinant proteins, antibiotics and genes. Development of new delivery systems that deliver the potential drugs spe-cifically to the target sites in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve de-sired goal has led to the evolution of the multidisciplinary field Nano biotech-nology which involves the combination of two most promising technologies of 21st century - biotechnology and nanotechnology.

2. What is nanotechnology?

It is the research and technology development at the atomic or macromolecu-lar levels. In the length scale of approximately 1 to 100 nm ranges, creating and utilizing structure devices, which have novel properties and functions because of their small or intermediate size, ability to control or manipulate of the atomic scale (1). The scale of some of the biologicals is as following.

Objects S i z e

Width of hair 50,000nm

Red Blood Cells 7,000nm

Bacteria 1,000n

Virus 100nm

Width of DNA 2.5nm

Aspirin Molecule 1 n m

2. 1. Nanobiotechnology

Nanotechnology applied to life sciences, biological components of living cells, deals with biomolecules in nanoscale (2, 3).

Nanoparticles in drug targetting

Shyamkumar Immadi*

Bonn-Aachen International Center for Information Technology B-IT, DahlmannstraBe2, D-53113 Bonn, Germany.

Received on:10-11-2011; Revised on: 15-12-2011; Accepted on:12-01-2012

ABSTRACT

Designing drug delivery systems is challenging in terms of targeting drugs to specific sites. Certain chemicals or therapeutics agents that show success in cell culture fail to produce same effects in the human body because of the limitation in targeting to the designated area. As a result, high concentrations are given to patients, resulting into more side effects. This case is similar to biotechnological drugs such as recombinant proteins, antibiotics and genes.Development of new delivery systems that deliver the potential drugs specifically to the target sites in order to meet the therapeutic needs of the patients at the required time and level remains the key challenge in the field of pharmaceutical biotechnology. Developments in this context to achieve desired goal has led to the evolution of the multidisciplinary field Nano biotechnology which involves the combination of two most promising technologies of 21st century -biotechnology and nanotechnology.

Key words: Nanoparticles in Drug targetting

Figure 1 Multi disciplinary functions of nanocarriers (4). 2.2. Nanocarriers

Multifunctional materials interact with biological system in well controlled way. They exhibit unique properties and functions because of their small size (2). Such nanostructures include liposomes, polymeric nanoparticles, solid lipid nanoparticles, dendrimers, functionalized nanocarriers, carbon nano structures; few examples of carbon nanostructure are buckyballs (fullerenes), nanotubes and nanowires.

Nanocarriers offer number of advantages making them ideal drug delivery vehicles. These include (4, 5).

♦ Nanocarriers can better deliver drugs to tiny areas within the body.

♦ Nanotechnology is so complimentary to biotechnology that prom-ises to bridge the gaps between the structure and the function of biomolecules as well as human physiology and pathophysiology.

♦ It is poised to help alleviate the problems of drug delivery with the development of nano structured delivery methods in combination with utilization of principles and techniques of biotechnology to manipulate molecular, genetic and cellular processes leading to the creation of a new inter disciplinary approach.

♦ It involves overlap of biotech, nanotech and information technol-ogy which is useful in gene therapy, drug delivery imaging, biomarkers, biosensors and novel drug discovery techniques.

♦ Nanocarriers overcome the resistance offered by the physiological barriers in the body because efficient delivery of drugs to various parts of the body is directly affected by particle size.

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♦ Nanoparticles aid in efficient drug delivery to improve aqueous solubility of poorly soluble drugs that enhance bioavailability of timed release of drug molecules and precise drug targeting.

♦ The surface property of nanocarriers can be modified for targeted drug delivery. For example, small molecules, proteins, peptides and nucleic acid loaded nanoparticles are not recognized by im-mune system and efficiently targeted to particular tissue types.

♦ Targeted nano-drug carriers reduce drug toxicity and provide more efficient drug distribution.

♦ Nanocarriers hold promise to deliver biotech drugs over various anatomic extremities of body such as blood brain barrier, tight epithelial junctions of the skin.

♦ Nanocarriers better penetrate tumors due to their leaky constitu-tion containing pores ranging from 100-1000nm in diameter. These carriers were discussed briefly in the following sections. 2.2.1. Liposomes

Liposomes are concentric bilayered vesicles in which an aqueous volume is entirely enclosed by a membranes lipid bilayer mainly composed of natural or synthetic phospholipids (4).These are formed when thin lipid films are lipid cakes are hydrated and stacks of liquid crystalline bilayers become fluid and swell. During agitation hydrated lipid sheet detach and self associated to form vesicles, which prevents interaction of water with the hydrocarbon core of the bilayer at the edges. The small unilamellar vesicles are surrounded by single lipid layer 25-50nm, where as several lipid layers separated by inter mutant aqueous layer surrounded by large unilamellar vesicles.

Fig 2:Liposomes (4).

Liposomes are characterized by in terms of size, surface charge and number of bilayers. It exhibits number of advantages in terms of ampiphilic charac-ters, bio compatibility and easy surface modification rendering it a suitable candidate delivery system for drugs (6).

2.2.2. Polymeric nanoparticles

Polymeric nanoparticles are nanosized colloidal structures composed of syn-thetic or semi synsyn-thetic polymers that vary in size 10-1000 nm. Polymeric materials exhibit several desirable properties, including biocompatibility, bio-degradability, surface modification and ease of fuctionalization of polymers. Polymeric systems have largely influenced the controlled and targeted drug delivery (4, 5).

Polymeric system allow for a greater control of pharmacokinetic behavior of loaded drug leading to more appropriate study levels of drugs. These at-tributes make it a candidate system for effective entrapment or encapsula-tion of biotech drugs that are usually sensitive to changes in the surroundings (4). Biodegradable polymeric nanoparticles typically consist of poly lactic

acid (PLA), poly glycolic acid (PGA), poly lactic glycolic acid (PLGA) and poly methyl methacrylate (PMMA) and are being investigated for the deliv-ery of proteins genes and DNA. PLGA based nano particles also proven their ability in enhanced delivery of antigen to dendritic cells. These are effective over all vaccine delivery system with an increased Ig-A antibody response (4). Delivery of antisense oligonucleotides and susceptible to deliv-ery of nucleus, polyalkylcyanoacrylate nanoparticles and nanocapsules were developed as peptide carriers for insulin. Polymeric nanoparticles bound peptides can be used for sustained oral delivery and also to improve absorp-tion and bioavailability. Water based polymeric biodegradable delivery sys-tem have been proposed for efficient delivery of peptides, Natural polymers like chitoson, gelatin, albumin, and sodium alginate have also been tried to toxicological problems associated with the use the synthetic polymers (6). Recently FDA approved drug made up of albumin nanoparticles is Abbraxane containing paclitaxel for us in patients with metastatic breast cancer who have failed combination therapy (5). Another work relating to chitosan nanoparticles NPs Toxaplasmagondii GRA1 protein and DNA vaccine were loaded on to chitosan NPs and result demonstrated its role in efficient oral delivery important class of biotech drugs for which polymeric nanoparticles offer an attractive possibility of vaccines protein, peptides, and oligonucle-otides delivery (4).

2.2.3. Solid lipid nanoparticles (SLN)

Solid lipid nanoparticles made from solid lipids are sub micron colloidal carriers 50-100nm dispersed either in water or in aqueous surfactants solu-tion (4, 6).

Different delivery routes have been exploited such as parenteral, and topical. Solid lipid nanoparticles are non toxic when compare with polymeric nanoparticles. Cationic solid lipid nanoparticles can serve as an effective, potent non viral transfection agent. SLN offers futuristic approaches as an effective adjuvant for vaccine to give a maximum immune response by opti-mizing surface properties. In a recent work, chitosan coated lipid nanoparticles for oral salmon calcitonin delivery were prepared which can be used for oral administration of peptidal drugs (4).This also serves as an effective alterna-tive to existing systems in terms of cost effecalterna-tive industrial scale- up tech-niques such as high pressure homogenization and micro emulsion technol-ogy, thereby paving its way to pharmaceutical industry (6).

2.2.4. Polymeric micelles

Fig 4: Polymeric micelles (4).

These systems include amphiphilic block copolymers such as pluronics (polyoxyethylene polypropylene block copolymers) that self associate in aqueous solution to form micelles. These are characterized by size and sur-face properties (7).

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stability in physiological solution leading to their slow dissolution in vivo (4, 7). Because of their core shells structure, these serve as suitable carrier for water insoluble drugs such drugs partition in hydrophobic core of micelles and outer hydrophilic layer aids in dispersion of aqueous media making it an appropriate candidate for intravenous administration (4, 7). Nanometric size range helps micelles to evade the reticulo endothelial system (RES), and aid passage through endothelial cells. Polymer micelles have been extensively studied as drug carriers. Conjugating to ligands such as antibodies can en-hance targeting potential of micelles (4, 7).

2.2.5. Functionalized nano carriers

The combination of functionalities of biomolecules and non-biologically de-rived molecular species used for special functions such as markers for re-search in cell and molecular biology, biosensing and bioimaging and marking of immunogenic moieties to targeted drug delivery are known as functionalized nano particles (4, 6).

These carriers are monodispersed sized particles of uniform shape with well defined surface composition. Nanoparticles from catalytically active metals such as platinum, palladium, silver and noncatalytically active metals such as gold have been prepared for functionalized purposes (4, 6). Organically functionalized nano particles of catalytic active metals offer a high surface area and unique size dependent chemical behavior. One approach is bioconjugated quantum dots as fluorescent biological labels. Quantum dots are crystalline clumps of several hundred atoms with an insulating outer shell of different material. Quantum dots can be attached to the biologicals such as cells, proteins and nucleic acids (6).

These can be constructed to emit the light at different wave lengths extending from ultraviolet to infra red range. Light emitted by these quantum dots is so bright that is possible to detect even the cellular and sub cellular structures. Quantum dots being of inorganic origin make the system stable, and their inert coating at inert surfaces makes them less toxic than dyes of organic origin. Thus quantum dots serve as an effective delivery and diagnostic agent to detect quantitatively the cellular contents (6).

2.2.6. Carbon nano structures

Different types of solid carbon structure were possible to be synthesized which can be used in nanobiotechnology. These were discussed in the following sections.

2.2.6.1. Carbon nanotubes

Elongated fullerenes resemble graphite sheets wrapped into cylinders. Length to width ratio is very high (few nano meters in diameter up to one nanometer in length) (8).

Fig 5. Carbon nanotubes (8). 2.2.6.2. Bucky balls (Fullerenes)

Buckyballs are named after architect R. Buckiministerfuller to whom 1996 noble prize was awarded in chemistry for their discovery (9). C60 is most stable among spherical fullerenes and is symmetrical resembling soccer ball.

Fig 6:Bucky balls (9). Fig 6:Bucky balls (9).

Some properties of carbon nano structures include high tensile strength, physically stability, superconductivity and they are chemically reactive with free radicals e.g. derivatives can be made more hydrophilic than fullerenes. New organic molecules can be generated by placing other atoms inside its cage (doping with alkali metals) (8).

Fig 7: Dendrimers (4). 2.2.6.3. Dendrimers

Spherical polymeric molecules Series of chemical cells built on small core molecules Each cell is called as generation, made from a core and altering a layers of two monomers acyclic acid and diamines molecular structures has a form of a tree with many branches can serve nanodevices for delivery of therapeutics (6).

3. Reasons for special consideration of nanobiotechnological products 1. Rapidly growing area of science (6).

2. Anticipitated to lead the development of novel and sophisticated (possible complex) application in drug delivery system (8). 3. Private sector, academic sectors and federal agencies are

develop-ing sustained programmes in nanotechnology

4. Significant research dollars being invested in nanotechnology (9). 4. Limitations

Nanocarriers exhibits difficulty in handling, storage and administration be-cause of susceptibility to aggregation. It has unsuitability for less potent drugs. But the key areas of concern are related to its small size as nanocarriers, it can gain access to unintended environments with harmful consequences. E.g. it can cross nuclear envelope of the cells and cause unintended genetic damage and mutations (10).

5. Some of the biological applications of nanobiotechnology

Biological nanodevices based on Dendrimers or being developed with the potentials to recognize cancer cells and diagnose cause of cancer delivery of drug to target report location of tumor report outcome of therapy. 5.1. Fullerene (C60) immunoconjugates

The field of biomedicine offers a promising arena for new applications of fullerene materials. The discovery of water-soluble C60 derivatives can cross cell membranes and even produce transfection, has accelerated interest in using C60 for diagnostic and therapeutic medicine. Growth factors, cytokines

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and anti bodies have all been extensively studied for their abilities to deliver payloads to the surface and the cytoplasm of target cells (11).

Fig 8: Shematic representation showing the formation of the C60 immunoconjugate from C60-SPDP (11).

Fig 9:TEM images of (a) ZME-018 antibody and (b) C60–Ser–(ZME-018) Immunoconjugates. The scale is the same for both frames; scale bar length is 20 nm. The solid curved feature in the image is the lacy carbon grid material (11).

The antibody designated ZME-018 targets the gp240 antigen (also known as the high molecular weight melanoma-associated antigen, HMWMAA) found on the surface of >80% of human melanoma cell lines and biopsy specimens. This antibody has previously been extensively used in clinical imaging trials and for the delivery of toxins, cytokines and other therapeutic agents to melanoma cells in vitro and in vivo (11, 12). Immunoconjugates containing ZME-018 is rapidly internalized into melanoma cells in culture (11). 5.2. Fullerenes antioxidant properties

Free radicals are typically oxygen molecules with unpaired electrons created as a byproduct of normal oxygen consumption. Free radicals damage intrac-ellular molecules by breaking critical intramolecular bonds, leading to

exten-sive cell injury and cell death (13). A single circulating free radical in the cell can damage tens or even hundreds of enzymes, membranes, or DNA mol-ecules through a destructive chain reaction of chemical damage that cascades through a cell until it is ultimately quenched (13).

Reactive oxygen species (ROS) play an important role in several diseases such cancer, cardiovascular diseases etc. the antioxidant property of some of the fullerenes might be useful.

Vitamin E

SOD Mitochondria

produce excess ROS

Endogenous antioxidants overwhelmed by the massive amounts of ROS

ROS H2O

Critical cellular molecules are chemically destroyed O2

Fig 10: ROS generation and its critical cellular damage (13). Fullerene antioxidants bind and inactivate multiple circulating intracellular free radicals, giving them unusual power to stop free radical injury and to halt the progression of diseases caused by excess free radical production (13). Fullerenes provide effective defense against all of the principal damaging forms of ROS: hydrogen peroxide, hydroxyl radical (and related hydroperoxy radicals), and superoxide. C-60 fullerene has thirty conjugated carbon-carbon double bonds, all of which can react with a radical species. In addition, the capture of radicals by fullerenes is very fast – in fact too fast to measure – and is referred to as “diffusion controlled”, meaning the fullerene forms a bond with a radical every time it encounters one (13).

Numerous studies demonstrate that fullerene antioxidants work significantly better as therapeutic antioxidants than other natural and synthetic antioxidants, at least for CNS degenerative diseases (13).

Fullerene antioxidants can enter cells and modulate free radical levels, thereby substantially reducing or preventing permanent cell injury and cell death

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Journal of Pharmacy Research Vol.5 Issue 2.February 2012 Fig 12: Antioxidative mechanism of fullerenes (13).

Fullerenes can capture multiple electrons derived from oxygen free radicals in unoccupied orbitals associated with its external electron cloud (13). C Sixty believes there are four additional unique mechanisms that optimize fullerene antioxidant behavior:

When a radical attacks a fullerene and forms a bond, this process creates a very stable and relatively unreactive fullerene radical. Thus the fullerene molecule has been called a “radical sponge.”

• The energy level of the first empty fullerene orbital is almost precisely the same as the energy level of the electron orbital in superoxide. As a result, the electron can easily “hop” from superoxide to the fullerene orbital (13).

• There is evidence to suggest the fullerene antioxidant quenching process is catalytic. This would imply that, at least in the case of superoxide, one fullerene molecule can react with many superox-ides without being consumed (13).

• Fullerene antioxidants localize within cells to mitochondria and other sites where excess free radical production occurs in disease states (13).

5.3. Nanobiotechnology for gene delivery

Fig 13 : Formation and characteristic properties of polymeric micelles (7).

Fig 14: Formation of lactosylated PIC micelles incorporating siRNA (small interfering) through the electrostatic interaction between PEG-b-siRNA conjugates and polycations. The acid-liable bond between PEG [Poly ethylene glycol] and siRNA (7).

Nevertheless, the lack of appropriate carrier systems for the in vivo siRNA delivery remains a limitation for clinical applications (7). siRNA Polymeric micelles might be a useful candidate for siRNA Nanocarriers. A negatively charged siRNA can be incorporated into PIC micelles through the electro-static interaction with PEG-b polycation block copolymers. In recent study, PEG-b polycation possessing. (PEG-b-DPT) was found to be remarkably effective for the siRNA delivery (14). This unique structure of PEG-b-DPT may allow only the primary amino group to be involved in the PIC forma-tion, thereby maintaining a buffering capacity of the secondary amino group for the proton sponge effect (7, 14).

The PIC micelles of PEG-b-DPT/siRNA showed a significant gene silencing toward endogenous genes (e.g., Lamin A/C) even after a 30-min pre-incuba-tion in 50% serum. These properties of the PIC micelles offer a promising feasibility for in vivo siRNA delivery. Recently conjugated siRNA with lactosylated PEG, through an acid labile linkage of the ß-thiopropionate to obtain Lac-PEG-b-siRNA, followed by complexation with the PLL[poly(L-lysine) homopolymers to form the lactosylated PIC micelles (7). Such PIC micelles of Lac-PEG-b-siRNA/PLL are assumed to be internalized through the ASGP (asialoglycoprotien) receptor mediated endocytosis and thereafter exerts the siRNA activity (15).triggered by the cleavage of the ß-thiopropionate bond under intracellular low pH conditions (15).

5.4. Nanoarrays for ultra sensitive biodetection

Dip-pen nanolithography was used to pattern MHA (16-mercaptohexa de-canoic acid) into an array of 60 nm dots on an Au thin film. The MHA was then deprotonated so the features are negatively charged (16). Monoclonal antibodies to the HIV-1 p24 antigen were then immobilized on the dots by immersing the array in anti-p24 immunoglobulin G (IgG) solution for one hour (16, 19).

Demonstration of measurable amounts of HIV-1 p24 antigen in plasma from men with less than 50 copies of RNA per milliliter illustrates that nanoarray-based assays can far exceed the 5 pg/ml detection limit of conventional enzyme-linked immunosorbent assays and provide sensitivity comparable to a polymerase chain reaction based assay, without target amplification This is the first clinical use of a nanoarray in biodetection with real patient samples (16). It could enable HIV-1 diagnosis in mother-to-child transmis-sion, where small sample volumes and immune complexes of passively trans-ferred maternal antibodies present challenges (16).

5.5. Nanobiotechnology for combination of drug design and drug delivery Potential applications of nanotechnology to facilitate drug delivery can be taken into consideration at the stage of drug design. A carrier nano particle can be designed simultaneously with the therapeutic molecule. An example is the approach of Calando Pharmaceuticals (Duarte, CA, USA), to design short interfering RNAs (siRNA) as anticancer agents using nano particle delivery (2, 3). Proprietary technology uses sequence selection and construction of effective siRNA molecules that bind to (and self-assemble with) the siRNA to form uniform colloidal-sized particles ~50 nm in diameter. As they are administered by intravenous injection, larger particles cannot get out of the blood and penetrate the tumor. If the drug particles are smaller than 10 nm they are quickly excreted through the kidneys (3, 20).

6. Potential challenges for nano drugs for bringing medicines to mar-ket

In addition to the generals aspects for evaluating traditional drugs the rela-tively new nano drugs may have to go through certain additional evaluation parameters (17, 18).

1. Are the methods used for characterization the chemical entity adequate and has the entity been adequately characterized to ensure good quality?

2. Are the efficacy characterization protocols adequate?

3. Are there any special safety and toxicity issues associated with size of nano drugs?

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Source of support: Nil, Conflict of interest: None Declared

4. Are there environmental issues associated with the nano formula-tion that should be addressed? This may not be as great as issue with drugs and devices because of the relatively small quantities of products when compared with typical consumer products (17, 18).

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