Targeted nanomedicines: effective treatment ... - Future Medicine

Review Targeted nanomedicines: effective treatment modalities for cancer, AIDS and brain disorders Novel technology in the nanomedicine field is expected to develop innovative products as targeted drugdelivery approaches. Targeted drug delivery of various drugs for the treatment of cancer, AIDS and brain disorders is the primary research area in which nanomedicines have a major role and need. This review is concerned with emerging targeted nanomedicines (polymeric nanoparticles, solid lipid nanoparticles, polymeric micelles, dendrimers, liposomes, gold nanoparticles and magnetic nanoparticles) and multifunctional carriers capable of combining targeted drug delivery and imaging (polymeric micelles, dendrimers and magnetic nanoparticles) in the field of pharmaceutical applications. The significant toxicity issues associated with these nanomedicines are also explored here. keywords: multifunctional nanomedicines, nanomedicines, targets, targeted drug delivery, toxicity

Madaswamy S Muthu1 & Sanjay Singh2† Author for correspondence: Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India 2 Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi - 221005, India Tel.: + 91 542 231 5871; Fax: + 91 542 236 8428; E-mail: drsanjaysingh@ rediffmail.com †

Nanomedicines, the medical applications of nanotechnology, are promising candidates for targeted drug delivery. Novel targeted drug-delivery approaches using nanomedicines are changing the future of therapy [1,2] . Developments in novel drug-delivery systems have facilitated the targeting of specific molecular targets for various therapies. Using the advancement of nanomedicines, these targets are now becoming specific organelles within individualized cells [3] . The most advanced nanomedicines are multifunctional nanomedicines, capable of simultaneously diagnosing and targeting drug to specific molecular targets by incorporating active molecules, targeting ligands and imaging agents [4] . The National Nanotechnology Initiative defines nanotechnology as the ‘understanding and control of matter at dimensions of roughly 1–100 nm, where unique phenomena enable novel applications’, enabling fabrication of devices on the nanoscale [101] . By contrast, Albert Franks defined nanotechnology as ‘that area of science and technology where dimensions and tolerances are in the range of 0.1 to 100 nm’ [1] . Although primary nanotechnology defines the size of the nanoparticles in nanomedicines between 1 and 100 nm in diameter, the size of the individual particles tested for drug delivery of therapeutic agents may range from 10 to 1000 nm [101] . Recently, attempts have been made to introduce a comprehensive definition for nanoparticles in nanomedicines [5] . For pharmac eutical purposes: ‘Nanoparticles are solid colloidal particles ranging in size from 10

to 1000 nm (1 µm), they consist of macromolecular materials in which the active principle (drug or biologically active material) is dissolved, entrapped, encapsulated and/or to which the active p rinciple is adsorbed or attached’ [6] . Particles larger than 200 nm can activate the human complement system and can be cleared from the blood by Kupffer cells. Additionally, splenic filtration captures particles that exceed slit size (200–250 nm) and liver filtration captures particles greater than 150 nm. Also, tumor capillaries rarely exceed 300 nm in diameter [7] . For these reasons, current research focuses on nanoparticle size of less than 200 nm. Even though, in some cases, nanomedicine containing particles are of less than 100 nm, some, such as magnetic nanoparticles, may accumulate in substantial amount in the reticuloendothelial system (RES) system [8] . The outer surface of most recently developed nanomedicines for targeted drug delivery are modified by attachment of various ligands onto the surface (i.e., specific antigens, antibodies and receptor targeting ligands) to facilitate targeted delivery to specific targets [9] . Although development of efficient nanomedicines extends into all therapeutic classes of pharmaceuticals, the development of effective treatment modalities for cancer, AIDS and brain disorders remains a therapeutically significant need. The information regarding the use of targeted nanomedicines for effective treatment of these diseases has not been well classified and documented. However, there are many reviews on specific nanomedicines [8,9] .

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Literature survey also indicates that limited attention has been given to long-term toxicity investigations of these nanomedicines developed for targeted drug delivery. Some toxicological reports have suggested that administration of nanomedicines leads to serious effects on biological systems [10] . This review, therefore, was conducted with the view to summarize the recent developments in the area of targeted nano medicines, for the effective treatment of cancer, AIDS and brain disorders, as well as the issues regarding their safety and toxicity. Targeting methods Targeting active molecules to specific sites in the body had been pursued actively ever since Ehrlich first envisaged the use of ‘magic bullets’ for the therapy of various diseases [11] . Interest in this concept has increased significantly in recent decades with the innovations of nanomedicine. Progress in the development of nanomedicines for targeted drug delivery has been reviewed by Moghimi and colleagues [7] . Targeted delivery can be achieved by either passive or active targeting.

Passive targeting Passive targeting is achieved by loading drug into a nanocarrier that reaches the target organ passively. Passive targeting of tumors takes advantage of hyper-permeable cells owing to their rapid vascularization. This rapid vascularization results in leaky, defective cells and impaired lymphatic drainage. Nanoparticles ranging from 10 to 100 nm then begin to accumulate within tumors because of their ineffective lymphatic drainage. This results in a phenomenon known as the enhanced permeation and retention (EPR) effect. The size and surface properties of a nanomedicine is vital for passive targeting. Nanoparticle size must be less than 200 nm to avoid uptake by the RES and its surface should be hydrophilic to avoid clearance by macrophages [12] . Active targeting Recent advances have led to the transformation from passive to active targeting. Active targeting of a drug is achieved by conjugating a nanocarrier system (drug loaded) to a tissue- or cell-specific targeting ligand. Active targeting has raised the importance of nanomedicine and this can now be achieved by a number of specific interactions, such as ligand–receptor and antibody–antigen binding. These specific interactions result in preferential accumulation of nanomedicine into molecular targets [13] . 106

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Specific targets in cancer Conventional cancer chemotherapies have doserelated side effects owing to nonspecific biodistribution of drugs. Targeted nanomedicines are emerging as one of the promising approaches in anticancer treatment and have major advantages. Cancer nanomedicines have the ability to improve the therapeutic index of drugs by preferential localization at target sites, lower distribution in healthy tissues, delivery of hydrophobic drugs and extended release rate [14] . By active targeting, the nanomedicine can deliver the anticancer drug specifically to malignant cells with the help of different targeting ligands attached to the nanomedicine. These ligands will bind to the specific receptors on the malignant cells. The different targeting ligands such as antibodies (Herceptin®, Mabthera® and Erbitux®, are antibodies that recognize HER2/ neu, CD20 and EGFR receptor, respectively), small molecules (folic acid, whose receptor expressed on the surface of the tumor cells) or peptides (amino acid sequence [Arg-Gly-Asp], which bind to tumor αvβ3 integrin) are attached covalently to the surface of nanoparticles in the nanomedicine for active targeting of anticancer drug to the malignant cells [15–19] . In the literature, antibody-carrying nanomedicines, such as immunodendrimers [20] , immunoliposomes [21] , immunopolymeric nanoparticles [22] and immunopolymeric micelles [23] , are also mentioned for active targeting. After the targeted nanomedicine attaches to the malignant cells, the drug will be released. Extending of the rate of release of the drug from the targeted nanomedicine will prolong the duration of action of the drug at target sites and reduce the adverse effects and, finally, will improve patient compliance [9] . Specific targets in AIDS The cells of the mononuclear phagocyte system, particularly monocytes/macrophages, have a key role in HIV infection and the immunopathogenesis of AIDS. They serve as a reservoir for the virus. Targeting of anti-HIV agents to these cells could improve their safety and efficacy [24] . Targeted nanomedicines can protect from HIV transmission as well as from its replication [24,25] . Some nanomedicines are designed as a targeted drug-delivery system to bind specifically to the gp120 proteins on the surface of HIV, through which the virus normally attaches to CD4 receptors on healthy cells. It protects against HIV transmission by occupying gp120 proteins [25] . RNA is the genetic material of HIV. Targeted future science group

Effective treatment modalities for cancer, AIDS & brain disorders

nanomedicine that binds specifically to TAR RNA of HIV and blocks binding by Tat protein, which is essential for HIV replication, would inhibit HIV replication during AIDS therapy [26] . Specific targets in brain disorders The BBB is the specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. Unlike peripheral capillaries that enable relatively free exchange of substances across/between cells, the BBB strictly limits transport into the brain through both physical (tight junctions) and metabolic (enzymes) barriers. Thus, the BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain [27] . Nanomedicines (such as polymeric nanoparticles and solid lipid nanoparticles) have become a most suitable option for targeted drug delivery into the brain because they can cross the BBB by various targeting mechanisms (i.e., enhanced retention of nanoparticles in the brain–blood capillaries and opening the tight junction of brain endothelial cells) [28,29] . Different targeted nanomedicines

Polymeric nanoparticles Polymeric nanoparticles are solid, colloidal particles consisting of macromolecular substances that vary in size from 10 to 1000 nm. The term

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nanoparticle is a collective name for both nanospheres and nanocapsules. Nanospheres have a matrix type of structure. Drugs may be absorbed at the sphere surface or encapsulated within the particle (Figure 1A) . Nanocapsules are vesicular systems in which the drug is confined to a cavity consisting of an inner oily core surrounded by a polymeric membrane. In this case, the active substances are usually dissolved in the inner core but may also be adsorbed to the capsule surface [30] . Polymeric nanoparticles from biodegradable and biocompatible polymers have been studied extensively as particulate carriers in the pharmaceutical and medical fields. The biodegradable polymeric nanoparticles have an advantage over liposomes through their increased stability and their unique ability to create an extended release. Polymeric nanoparticles are used most in medical applications and have attracted considerable attention as targeted drug-delivery carriers owing to their biocompatibility, physical stability, protection of incorporated labile drugs from degradation and controlled release [31,32] . Following oral administration, polymeric nanoparticles of less than 500 nm can cross the M cells in the Peyer’s patches of the intestine and particles are taken up by the lymphatic system. Transport of such drugs through the intestinal lymphatics to systemic circulation avoids presystemic hepatic metabolism and therefore enhances the bioavailability of drugs [33,34] . These carriers have been investigated especially in drug-delivery

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Figure 1. Different surface unmodified nanomedicines. (A) Polymeric nanoparticles; (B) solid lipid nanoparticles; (C) polymeric micelles; (D) dendrimers; (E) liposomes; and (F) magnetic nanoparticles.

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systems for drug targeting because their particle size (ranging from 10 to 1000 nm) is acceptable for intravenous injection. The clearance mechanism of intravenous administered surface unmodified nanoparticles is the spontaneous adsorption of plasma protein (opsonization process), which are capable of interacting with the plasma membrane receptors on monocytes and macrophages, thus promoting particle recognition. In the next stage, phagocytosis of the nanoparticles by the circulating macrophages leads to their simultaneous elimination from the systemic circulation and accumulation mainly in liver [7] . Opsonization of polymeric nanoparticles can be prevented by providing a surface of the particles with a hydrophilic group (stealth nanoparticles) (Figure 2A) . Thus, if the carrier size is under 1 µm, an intravenous injection (the diameter of the smallest blood capillaries is 4 µm) is enabled and this carrier size is also desirable for intramuscular and subcutaneous administration, minimizing any possible embolism [35–37] . Albumin-bound paclitaxel (Nab™–paclitaxel), approved by the US FDA as a nanomedicine, is a novel toxic solvent-free nanomedicine (polymeric nanoparticles) used for metastatic breast cancer. Nab–paclitaxel has an advantage over Cremophor ®-EL paclitaxel in that it avoids the hypersensitivity reaction associated with Cremophor-EL, the solvent in traditional paclitaxel, and uses the endogenous albumin-transport mechanisms (passive targeting) to concentrate paclitaxel within the tumor cells. Nanoparticles

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Figure 2. Different stealth nanomedicines for passive targeting. (A) Polymeric nanoparticles; (B) solid lipid nanoparticles; (C) liposomes; and (D) magnetic nanoparticles.

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in this nanomedicine have sizes of approximately 100 nm and offer the ability to solubilize paclitaxel without using any toxic organic solvent, which enabled increasing administered doses, thus increasing overall drug efficacy. The reported side effects of Nab–paclitaxel include hair loss, infection owing to low white blood cell count, fatigue and weakness, low red blood cell count, mouth or lip sores, joint and muscle pain, stomach upset, diarrhea and cardiovascular effects [38] . Doxorubicin was incorporated into biodegradable acrylate nanoparticles of polyisohexylcyanoacrylate. This resulted in an increase in cytotoxicity and a reduction in cardiotoxicity in preclinical studies. During clinical studies on refractory solid tumors at six dosing regimens (15, 30, 45, 60, 75 and 90 mg/m 2 ), no cardiotoxicity was reported. However, the doselimiting toxicity observed was neutropenia and hematological toxicity at higher doses (75 and 90 mg/m2). Presently, a doxorubicin polymeric nanomedicine is under Phase II trials [39] . Herceptin and Mabthera (cancer cell-targeting antibodies) were coupled covalently to biodegradable polymeric nanoparticles for active targeted delivery. Poly(d,l‑lactic acid) nanoparticles with a mean size of approximately 170 nm were prepared and covalent coupling of the antibodies to thiolated nanoparticles was obtained through a bifunctional cross-linker. The smaller size of the polymeric nanoparticles provided a longer circulation halflife and increased the chance of reaching the target site (Figure 3A). The specific interaction as well as cellular localization of Herceptin nanoparticles and Mabthera nanoparticles in SKOV‑3 (containing HER2/neu receptor) and Daudi (containing CD20 receptor) cancer cells, respectively, were studied. These antibody-labeled nanoparticles represent a promising approach for polymeric nanoparticles in active targeting for cancer therapy [22] . HIV‑1 Tat (DNA) represents a relevant antigen for the development of a prophylactic and/ or therapeutic vaccine against AIDS. Recently, poly(methylmethacrylate)-based polymeric nanoparticles efficiently adsorbed HIV‑1 Tat (antigen) molecules, mainly through electrostatic interactions, preserved its conformation and activity, delivered it into the cells in the absence of any in vitro or in vivo cytoxicity, increased the shelf-life of HIV‑1 Tat and produced the antigen-specific cellular responses [40] . Chen and colleagues have reviewed polymeric nanoparticles as suitable delivery systems for brain targeting owing to their enhanced retention future science group


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Figure 3. Different surface-modified nanomedicines using targeting ligand/molecules for active targeting. (A) Polymeric nanoparticles; (B) solid lipid nanoparticles (thiamine coated); (C) polymeric micelles; (D) dendrimers; (E) liposomes; (F) gold nanoparticles; and (G) magnetic nanoparticles.

in the brain–blood capillaries [28] , with adsorption onto the capillary walls, resulting in a high concentration gradient across the BBB, opening of tight junctions owing to the presence of nanoparticles and transcytosis of nanoparticles through the endothelium. Furthermore, coating of these polymeric nanoparticles with polysorbate has been reported to improve brain targeting by facilitating an interaction with the BBB endothelial cells. The effects observed owe to their solubilization of endothelial cell membrane lipids and membrane fluidization, surfactant effects of polysorbates, endocytosis of polymeric nanoparticles and inhibition of efflux system [41] . An attempt was made to target the anti-Alzheimer’s drug rivastigmine in the brain using poly(n‑butylcyanoacrylate) nanoparticles. The drug was administered as free drug, bound to future science group

nanoparticles and also bound to nanoparticles coated with polysorbate 80. In the brain, a significant increase in rivastigmine uptake was observed in the case of poly(n‑butylcyanoacrylate) nanoparticles coated with polysorbate 80 compared with the free drug [42] . Most studies in the literature are related to targeted drug delivery to brain by using polysorbate 80 coated polymeric nanoparticles. Little is mentioned about the safety and toxicities of using polysorbate 80 as surfactant when formulated together with polymeric nanoparticles [43] . Toxicities have become even more serious for targeted nanomedicines during intravenous administration because their size partly determines tissue distribution. Thus, the fate of nanomedicines and their constituents in design and particularly those that are not biodegradable, such as coating agents, like polyethylene glycol www.futuremedicine.com

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(PEG), should be investigated [44] . In one study, degradation products produced by poly(l-lactic acid) particles showed cytotoxicity. During the injection of these particles into the peritoneal cavity of mice, macrophages demonstrated signs of cell damage, cell death and cell lysis owing to phagocytosis of a large amount of poly(l‑lactic acid) particles [45] . Pluronics used for surface modification and design of nanomedicines act as inhibitors of P‑glycoprotein efflux pumps expressed in polarized endothelial cells that form the exterior of the BBB and could potentially interfere with the transport of a number of modulators and homeostatic mediators in the brain [46] .

Solid lipid nanoparticles Solid lipid nanoparticles (SLNs) are an alternative colloidal matrix carrier system for targeted drug delivery (Figure 1B) . Compared with polymeric nanoparticles, SLNs have more advantages for drug-delivery systems, such as good tolerability, biodegradation and high bioavailability by targeting effect [47] . It is useful for lipophilic drugs, such as clozapine (needed for brain targeting), to avoid first-pass metabolism [48] . Highly lipophilic compounds, such as long-chain triglyceride lipids (trimyristin, tripalmitin and tristearin), reach systemic circulation through the lymphatics. The majority of fatty acids, with chain lengths of 14 and above, were recovered in thoracic lymph. These triglycerides are suitable for preparing lymphatic-targeting SLNs and to improve the bioavailability of the drugs. Transport of drug-loaded SLNs through the intestinal lymphatics through thoracic lymph duct to systemic circulation joining at the junction of the jugular and left subclavian vein avoids presystemic first-pass metabolism and therefore enhances the bioavailability of the drug [33] . SLNs of below 200 nm in size have an increased blood circulation and thus an increase in the time for which the drug remains in contact with the BBB and for the drug to be taken up by the brain. These carriers can gain access to the blood compartment easily (because of their small size and lipophilic nature). Opsonization of SLNs can be prevented by coating the particles with a hydrophilic or a flexible polymer and/or a surfactant. Targeting ligands that specifically bind to surface epitopes or receptors on the target sites can be coupled to the surface of SLNs for targeting the brain (through the BBB) [49,50] . Jain and colleagues fabricated ferritin-coupled SLNs and investigated tumor-targeted delivery of 5‑flurouracil (anticancer drug) [51] . Ferritin 110


used as targeting ligands that specifically bind to surface epitopes or receptors on the tumor cells for effective reduction in tumor growth. Permeability of stavudine, delavirdine and saquinavir across the BBB was investigated by incorporating these into SLNs. Through the BBB model, anti-HIV drug-loaded SLNs were demonstrated as targeting colloidal drug-delivery systems [52] . Zara and colleagues made nonstealth and stealth SLNs of doxorubicin [53] . They used PEG at various concentrations as the ‘stealthing’ agent. The intravenously administered SLNs and stealth SLNs containing increasing amounts of stealthing agent, enabled doxorubicin loaded nanoparticles to be transported through the BBB. They observed an increase in the brain concentration of doxorubicin on increasing the stealthing agent (Figure 2B) . Reddy and Venkateshwarlu studied brain levels after intravenous administration of etoposideloaded tripalmitin nanoparticles and etoposide solution [54] . The authors found a relationship between the charge on the SLN and the brain drug levels. Allen and colleagues proposed a design of SLNs attached with specific ligand (thiamine ligand coating, which prefers to bind to the blood thiamine transporters for BBB targeting) on the surface of SLNs in the size of 100 nm, which could lead to their increased retention at the BBB and a consequent increase in nanoparticle concentration at the surface of the BBB [29] . The blood-retention properties of these nanoparticles may increase their potential utility in targeting and controlled drug delivery (Figure 3B) .

Polymeric micelles Polymeric micelles have several advantages over conventional surfactant micelles in that they have better thermodynamic stability in physio logical solution, as indicated by their low critical micellar concentration, which makes polymeric micelles stable and prevents their rapid dissociation in vivo. Micelles have a fairly narrow size distribution in the nanometer range and are characterized by their unique core-shell architecture, in which hydrophobic segments are segregated from the aqueous exterior (Figure 1C) [55] . Many existing solvents for poorly water soluble pharmaceuticals, such as Cremophor EL or ethanol, can be toxic, which limits therapeutic doses and restricts treatment options. Polymeric micelles provide a safer alternative for parenteral administration of poorly water-soluble drugs. Drugs can be partitioned in the hydrophobic core of micelles and the outer hydrophilic layer forms future science group


a stable dispersion in aqueous media, which can then be administered intravenously. The distribution of drug-loaded polymeric micelles in the body is determined mainly by size and surface properties. Their individual particle size is less than 50 nm in diameter, which provides obvious benefits over liposomes. It makes them ideal drugdelivery carriers because they avoid renal exclusion and the RES but also provides them with enhanced endothelial cell permeability in the vicinity of solid tumors by passive diffusion [55–57] . Targeted drug delivery by polymeric micelles is, in most cases, hindered by either premature drug release from the micellar nanomedicine before the nanomedicine reaches the specific targets [58] . As with other nanomedicines used for targeted drug delivery, the drug-delivery potential of polymeric micelles may be enhanced by conjugating targeting ligands, including antibodies to the micelle surface. Recently, Torchilin and colleagues have formulated antitumor antibodyconjugated polymeric micelles (immunomicelles), encapsulating water-insoluble drug taxol inside the hydrophobic core of the micelles (Figure 3C) [59] . Micelles were formulated using PEG-phosphatidylethanolamine (PEG-PE) with the addition of the small fraction of p‑nitrophenylcarbonyl-PEG-PE. The PE form the hydrophobic core of the micelles, whereas p‑nitrophenylcarbonyl enables efficient attachment of an amino group containing antibodies by the formation of the urethane bond. They found that such immunomicelles are effectively recognized and bound to various cancer cells in vitro. They demonstrated that, compared with nontargeted micelles, immunomicelles were capable of delivering higher concentrations of drugs to tumors in mice by an active targeting method. Depending on the nature of monomers used in the preparation of polymeric micelles, these can induce cell death by apoptosis, necrosis or both. Differential gene expression has been reported in certain cells after cisplatin delivery with polymeric micelles when compared with that of free cisplatin treatment. Cisplatin micelles downregulated the gene expression of integrin and matrix metalloproteases families, whereas free cisplatin upregulated them. The results suggest that differential gene expression may lead to additional therapeutic effects of c isplatin micelles [60–62] .

Dendrimers Dendrimers are macromolecular nanoparticles that comprise a series of branches around an inner core (Figure 1D) . Dendrimers used in targeted drug delivery are usually 10 to 100 nm. future science group

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Dendrimers can be synthesized starting from the central core and working out toward the periphery (divergent synthesis) or in a top-down approach starting from the outermost residues (convergent synthesis) [63] . Structure of these polymers is repeated branching around the central core that results in a nearly-perfect 3D geometrical pattern. At higher generations, dendrimers resemble spheres, with a number of cavities within its branches to hold both drug and diagnostic agents. In theory, it is possible to synthesize amphiphilic dendri mers with a hydrophobic core inside hydrophilic branching [63,64] . Dendrimers are useful antiviral agents in that they enable the presentation of multiple ligands on a single molecule as a result of a high number of pharmacophores. Because of its size and multivalent nature, a dendrimer can bind to many receptors simultaneously as compared with other single molecules, which can interact with only one receptor; therefore, these multivalent dendrimers can enhance biological effects [65] . Dendrimers may be toxic because of their ability to disrupt cell membranes as a result of a positive charge on their surface. In some cases, a dendrimer-encapsulated drug for targeted drug delivery tends to release rapidly, before it has reached a target site [66] . A study was performed using fifth generation polyamidoamine dendrimers conjugated with HER2/neu antibody (Figure 3D) for the targeting of cancer cell receptors, which are often overexpressed in breast and ovarian malignancies. The conjugates showed binding and internalization into HER2/neu-expressing cells. Specific and increased binding to HER2/neu-expressing tumors was also demonstrated in vivo. The time course of internalization reveals a faster and more efficient internalization of the conjugate than antibody alone [20] . In another study, poly(propyleneimine) dendrimers loaded with efavirenz and conjugated with tuftsin were prepared to target human monocytes/macrophages. These dendrimers enhanced the cellular uptake of the drug-loaded carrier by stimulating the phagocytic activity of the monocytes/macrophages especially in HIVinfected cells and contributing to the anti-HIV activity of the drug owing to its inherent antiretroviral activity [67] . VivaGel® is a water-based gel with a polylysine dendrimer as its active ingredient. It has been developed by Australian nanotechnology company, Starpharma, for the prevention of HIV and non-HIV infection. Investigational new drug www.futuremedicine.com

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application (IND) for this novel dendrimer-based drug was submitted to the US FDA in June 2003 and the first clinical trial under this IND was completed in 2004. The drug was a microbicide designed for the prevention of HIV and sexually transmitted infections. Active groups on the surface of the dendrimers are designed to bind to the gp120 proteins on the surface of HIV, through which the virus normally attaches to CD4 receptors on healthy cells. Currently, Phase II trials are investigating VivaGel and have been granted Fast Track status by the FDA [25,68] . Zhao and colleagues prepared a polyamidoamine dendrimer for HIV targeting [26] . During an interaction study, dendrimers disrupted the interaction of Tat peptide with TAR RNA, which is essential for HIV‑1 virus replication. They suggested that binding processes of dendrimer-TAR RNA have great significance for the design of new drugs for the therapy of AIDS.

Liposomes The first suggested use of liposomes came from the group of Weismann in 1969 [69] . Liposomes are small artificial vesicles of spherical shape that can be produced from natural nontoxic phospholipids and cholesterol (Figure 1E) . Liposomes vary greatly in size, most are 400 nm or less. Because of their size, hydrophobic and hydrophilic character, biocompatibility, biodegradability, low toxicity and immunogenicity, liposomes are promising systems for drug delivery [70] . Liposome surfaces can be modified by attaching PEG units to the bilayer (producing what is known as stealth liposomes) to enhance their circulation time in the bloodstream (Figure 2C) . Doxil®, a long-acting PEGylated liposomal formulation of doxorubicin, is known for its significant improvements over doxorubicin as nanomedicine. Daunorubicin (Daunoxome®) is being marketed currently in liposome delivery systems, whereas vincristine (Onco TCSTM) awaits FDA approval [71–75] . However, there have been major drawbacks to the use of liposomes for targeted drug delivery. Some of the major problems are poor control over the release of the drug from the liposomes (i.e., the potential for leakage of the drug into the blood), stability, poor batch-tobatch reproducibility, difficulties in sterilization and low drug loading [70] . Similar to polymeric nanoparticles, polymeric micelles and dendrimers, liposome surfaces can be attached with targeting ligand (antibodies for immunoliposomes) molecules for active targeted delivery to specific tissues/cells (i.e., cancer cells) [76] . 112


Targeted delivery of immunoliposomes was examined for cancer therapy (Figure 3E) . Cancer cell-targeting antibodies (HER2/neu and CD20 antibodies) were conjugated on the liposomal surface. Antibodies were attached to the distal termini of PEG chains on sterically stabilized immunoliposomes to avoid interference from the PEG chains with its interaction with the HER2/ neu and CD20 receptor on the cancer cell surface. The efficacy of liposomes to targeted cancer cells was significantly higher [77] . HLA-DR is the most frequently identified host of HIV‑1 in tissues of HIV-infected individuals. Studies showed that the subcutaneous injection of liposomes bearing antibodies of HLA-DR fragments to mice resulted in an increased accumulation in lymph nodes when compared with nontargeted liposomes [78] . In another study, authors have demonstrated that HLA-DR antibody-loaded immunoliposomes containing indinavir (anti-HIV drug) were as efficient as the free agent at inhibiting HIV‑1 replication [79] . OX26 monoclonal antibody is transported across the BBB by receptor-mediated transcytosis. In one experiment, nonviral gene delivery using OX26 immunoliposomes was associated with a clear pharmacological effect resulting from reversible normalization of striatal tyrosine hydroxylase expression in a rat model of parkinsonism. These findings clearly demonstrate transcytosis of OX26 immunoliposomes across the BBB in vitro as well as in vivo [80] . Parenteral (intravenous) injection of some liposomes can cause acute hypersensitivity reactions in a high percentage (up to 45%) of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement system on the surface of lipid particles, leading to anaphylatoxin liberation and subsequent release reactions of mast cells, basophils and possibly other inflammatory cells in blood [81] . In one study, idiosyncratic reactions occur after infusion of stealth systems, such as PEG-grafted liposomes. Based on in vitro and animal studies, it was proposed that the phenomenon might represent an unusual allergic reaction called complement activation-related pseudoallergy [82] .

Gold nanoparticles Gold nanoparticles based on gold cores are promising candidates that provide many desirable values for drug-delivery systems. They can be prepared with core size from 1.5 to 10.0 nm, providing a large area for efficient drug loading future science group


and ligand conjugation. The drug loading can be made by either noncovalent interaction (e.g., DNA or enzymes through electrostatic interaction) or covalent chemical conjugation of drug [83–85] . Inherent features of gold nanoparticles are tunable core size, monodispersity, low toxicity, large surface to volume ratio and ease of fabrication and multifunctionalization. Human cells can take up these gold nanoparticles without any cytotoxic effects. In addition, gold nanoparticles are highly attractive, stable and versatile for targeted drug delivery [85] . Gold nanoparticles were examined for uptake and acute toxicity studies in human leukemia cells. The results suggested that some precursors of these nanoparticles may be toxic [83] . Targeted drug delivery of gold nanoparticles can be achieved through a transmembrane receptor-mediated endocytosis pathway. Gold nanoparticles can be conjugated with a drug and with a targeting ligand that specifically recognizes the target receptor for active targeting. Transferrin is a protein that can be used as a targeting ligand because many cancer cells overexpress transferrin receptors on their surface. Yang and colleagues have reported the enhanced uptake of transferring-conjugated gold nanoparticles by human nasopharyngeal carcinoma cells [86] . They demonstrated the transferrin receptormediated delivery of gold nanoparticles and confirmed the endocytosis process of nanoparticles by atomic-force microscopy (Figure 3F) .

Magnetic nanoparticles Magnetite (Fe3O4) and maghemite (γ‑Fe2O3) are the most commonly used magnetic nanoparticles for targeted drug delivery owing to their chemical stability and biocompatibility (F igur e 1F) . Inorganic magnetic nanoparticles can diffuse through biological membranes and interact closely with biomolecules [87] . For intravenous administration, they are coated with hydrophilic macromolecules to avoid their clearance by the opsonization process (Figure 2D) [88] . During active targeting, they are attached with targeting ligands (oligosaccharides, folic acid [Figure 3G] , antibodies and their fragments) that are expected to bind specifically to the surface receptors on the target sites [89] . A Phase I clinical study was reported using 14 cancer patients. The magnetic core nanoparticles were coated with starch particles (50–150 nm) containing anticancer drug. The treatment used with the magnetic nanoparticles in patients was well tolerated, evidence of the accumulation of particles in the tumor cells with low side effects [90] . future science group

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Novel applications of magnetic nanoparticles are expected in the field of cancer thermal treatment, magnetic targeting and remotely triggered targeted drug release [8] . There are also a few toxicity reports of inorganic magnetic nanoparticles at different levels. In one study, the lethal dose of the dextran–iron oxide complex and pristine iron oxide was reported after toxicity evaluation [8] . Multifunctional nanomedicines for target drug delivery Multifunctional nanomedicines can be designed to facilitate simultaneous active targeted drug delivery and imaging. Imaging or contrast agents are entrapped within the hydrophobic core or linked covalently to the surface of the multifunctional nanomedicines loaded with drug and with attachment of the targeting ligand. These nanomedicines may circulate for prolonged periods in the blood, evading host defenses and gradually release drug by targeting and simultaneously facilitate in vivo imaging [4] .

Polymeric micelles Recently, researchers fabricated multifunctional polymeric micelles based on amphiphilic block copolymers to incorporate doxorubicin (anticancer drug) and iron oxide nanoparticles (imaging agent) inside polymeric micelles that were then attached with the cancer cell targeting-specific marker αvβ3 integrin [91] . In another study by Yang and colleagues [92] , prepared multifunctional polymeric micelles of less than 100 nm in size that contain a molecular-targeting ligand (folic acid) on the micelle surface as well as a cluster of superparamagnetic iron oxide nanoparticles in the cores for magnetic imaging. Micelles based on copolymers of poly(ε-caprolactone) (PCL) and PEG-bearing folic acid on the PEG distal ends, denoted as folic acid–PEG–PCL, were used to encapsulate the anticancer drug doxorubicin and superparamagnetic iron oxide. They demonstrated their potential as multifunctional polymeric micelles that can transport anticancer drugs to tumor cells effectively (Figure 4A) . Dendrimers High density surface groups of dendrimers make this system ideally suited for multifunctional use of dendrimers. In a recent study, the primary amino groups on the surface of the fifth generation Poly(amidoamine) dendrimer were neutralized through partial acetylation, providing enhanced solubility of the dendrimer and www.futuremedicine.com

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preventing nonspecific targeting interactions during targeted delivery. An imaging agent (fluorescein isothiocyanate), targeting ligand (folic acid) and anticancer drug (paclitaxel) were conjugated to the remaining nonacetylated primary amino groups of a fifth generation dendrimer for targeted delivery to cancer cells (Figure 4B) . Biological in vitro testing has shown uptake and cytotoxic effect of this multifunctional dendrimers to specific cancer cells [93] .

Magnetic nanoparticles Magnetic nanoparticles can be used for the imaging of specific cellular events and biological activity. By adding a peptide/small molecule (targeting ligand) to the surface of iron oxide nanoparticles, they are able to accumulate in cancer cells [94] . Finally, drugs can be conjugated to the surface or encapsulated within a polymer shell surrounding the iron oxide core for targeting the drug and for imaging (multifunctional nanomedicines). Hu and colleagues [95] synthesized multifunctional tamoxifen-loaded Fe3O4/poly(l‑lactic acid) nanoparticles and evaluated its cytotoxicity against MCF‑7 breast cancer cells. Nanoparticles with an average size of nearly 200 nm were synthesized with superparamagnetic property provided

75 nm 100 nm

Polymer coating

Drug Hydrophilic group Targeting molecules

Fe3O4

200 nm

Imaging agent Magnetic particles


Figure 4. Different multifunctional nanomedicines. (A) Polymeric micelles; (B) dendrimers; and (C) magnetic nanoparticles (polymer coat loaded with anticancer drug).

114


by encapsulating Fe3O4 nanoparticles in the tamoxifen-loaded poly(l‑lactic acid) matrix. The superparamagnetic property of these nanoparticles means that they can also be used as contrast agents for MRI and the distribution of tamoxifen-loaded magnetitie/poly(l‑lactic acid) nanoparticles can be visualized in vivo. Moreover, nanomedicine formulated with tamoxifen can increase drug concentration in tumors through the EPR effect. Cytotoxicity assay shows that multifunctional tamoxifen-loaded magnetitie/poly(l‑lactic acid) nanoparticles exhibit no significant cytotoxicity against MCF‑7 breast cancer cells (Figure 4C) . Some selected targeted nanomedicines under progress currently for the treatment of cancer, AIDS and brain disorders are listed in Table 1. Conclusion Recent developments of nanomedicines have already developed some new, effective targeted drug-delivery systems for the better improved therapy of cancer, AIDS and brain disorders. Finding new molecular targets and knowledge of molecular pharmacology, physiology, biotechnology and nanotechnology will bring major inputs in the development of nanomedicines for targeted drug delivery. However, the toxicity issues of the developed nanomedicines must be investigated to prove their safe and efficacious use. Therefore, future design of safer nanomedicines should be based on the detailed and thorough understanding of biological processes. Future perspective In the next 5 years, research on nanomedicines in targeted drug-delivery systems will lead to breakthroughs that enable their therapeutic applications. Specifically, nanomedicines may avoid the need for risky administration of drugs, thereby promoting patient compliance and therapeutic effects. Targeted nanomedicines for cancer therapy may avoid the adverse effects (such as immunosuppression, cardiomyopathy and neurotoxicity) of traditional cancer therapies, while also providing improved therapeutic efficacy. Therefore, targeting cancer nanomedicines have developed rapidly. Targeted cancer therapeutics will be developed to ease administration and improve safety for patients. High architectural control characteristic of cancer-targeted nanomedicines will lead to positive outcomes from in vitro and in vivo studies. Development of targeted drug-delivery approaches for AIDS therapy will improve the safety and efficacy of anti-HIV agents by future science group


Review

Table 1. Some selected targeted nanomedicines currently under progress for the treatment of cancer, AIDS and brain disorders. Type of targeted nanomedicine

Specific target (disease)

Practical success

Polymeric nanoparticles

Promising approach for effective Preclinical cancer therapy Enhanced retention in brain Preclinical High concentration of nanomedicine Preclinical in tumor

3A

[22]

Solid lipid nanoparticles Polymeric micelles

HER2/neu and CD20/cancer BBB/brain Tumor cells/cancer

3B 3C

[29]

Dendrimers

HER2/neu/cancer

Increased receptor binding

Preclinical

3D

[20]

Dendrimers

gp120 of HIV/AIDS

Effective treatment by multiple receptor binding

Phase II trials

3D

[25]

Liposomes

HER2/neu and CD20/cancer Transferrin receptor/ cancer HER2/neu/cancer

Increased anticancer efficacy

Preclinical

3E

[77]

Enhanced specific uptake by carcinoma cells Enhanced accumulation of nanomedicine Enhanced transport to tumor cells

Preclinical

3F

[86]

Preclinical

3G

[89]

Preclinical

4A

[92]

Enhanced transport to tumor cells

Preclinical

4B

[93]

Enhanced uptake of drug and nanomedicine without significant cytotoxicity

Preclinical

4C

[95]

Gold nanoparticles Magnetic nanoparticles Multifunctional polymeric micelles Multifunctional dendrimers

Folic acid receptor/ cancer Folic acid receptor/ cancer

Multifunctional magnetic nanoparticles

MCF-7 breast cancer cells/cancer

reducing their dose and the adverse effects associated with them. The amazing growth in recent approaches towards a BBB-targeted drug-delivery system using nanomedicine will provide novel nanomedicines for brain disorders. Also, emerging nanotechnology will soon permit nanomedicine to fuse with oral, injectable, implantable and transdermal drug delivery systems for targeted therapy with simultaneous diagnosis (multifunctional nanomedicine). Prospective nanomedicines that have biosensing functionalities with in vivo feedback will develop ‘smart nanomedicines’ for drug delivery. Although these nanomedicines may offer various advantages over conventional drug-delivery systems, their safety should not be ignored. The toxicity of these nanomedicines may be due to their large surface area. A challenging direction for the

Level of development

Figure

Ref.

[59]

development of targeted nanomedicines is to avoid such pitfalls associated with these nanomedicines, for example, by better material selection and targeting ligand modifications. Future developments in gene-derived toxicogenomic research may create new methods for assessing toxic effects of targeted nanomedicines in biological systems. Financial & competing interests disclosure We acknowledge financial support from University Grant Commission, New Delhi, India, in terms of a Senior Research Fellowship. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Nanomedicines have drug targeting or multifunctional capabilities and are currently under intense development for therapeutic applications in cancer, AIDS and brain disorders. Nanomedicines have been developed for actively targeting various molecular targets of cancer, AIDS and brain disorders by different targeting mechanisms. Targeted drug delivery of nanomedicines can be achieved by either passive or active targeting. The toxicology of intravenously injected targeted nanomedicines and other material involved in their design can provide safety limits on the usage of nanomedicines. Multifunctional nanomedicines are capable of simultaneously diagnosing and targeting drugs to specific molecular targets by incorporating active molecules, targeting ligands and imaging agents.



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