Experimental and Molecular Pathology 86 (2009) 215–223
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Experimental and Molecular Pathology
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Review
Nanoparticle-based targeted drug delivery
Rajesh Singh a, James W. Lillard Jr. a,b,⁎a Department of Microbiology and Immunology, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202, USAb Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, USA
Abbreviations: BBB, blood–brain barrier; CPT, campethylene oxide–propylene oxide block copolymer; C60,receptor 2; MRI, magnetic resonance imaging; Mabs, moA; C32, polybutane diol diacrylate co amino pentanol; PCacid; PLGA, poly lactide-co-glycolide; Tween 80, polysor⁎ Corresponding author. James Graham Brown Cancer
The development of a wide spectrum of nanoscale technologies isbeginning to change the scientific landscape in terms of diseasediagnosis, treatment, and prevention. These technological innova-tions, referred to as nanomedicines by the National Institutes ofHealth, have the potential to turn molecular discoveries arising fromgenomics and proteomics into widespread benefit for patients.Nanoparticles can mimic or alter biological processes (e.g., infection,tissue engineering, de novo synthesis, etc.). These devices include, butare not limited to, functionalized carbon nanotubes, nanomachines(e.g., constructed from interchangeable DNA parts and DNA scaffolds),nanofibers, self-assembling polymeric nanoconstructs, nanomem-branes, and nano-sized silicon chips for drug, protein, nucleic acid,or peptide delivery and release, and biosensors and laboratorydiagnostics.
Biodegradable polymers have been studied extensively over thepast few decades for the fabrication of drug delivery systems.Considerable research is being directed towards developing biode-gradable polymeric nanoparticles for drug delivery and tissueengineering, in view of their applications in controlling the releaseof drugs, stabilizing labile molecules (e.g., proteins, peptides, or DNA)from degradation, and site-specific drug targeting. The late 1969s andearly 1979s saw the advent of polymer microparticles based onacrylamide micelle polymerization (Kreuter, 1994a). Since then, alongwith different polymerization methods, preformed polymers alsohave been developed and studied (Barratt, 2000; Kreuter, 1994a; Pittet al., 1981). The majority of studies on nanoparticles reported to datehave dealt with microparticles created from poly(D,L lactide), poly(lactic acid) [PLA], poly(D,L glycolide) [PLG], poly(lactide-co-glycolide)[PLGA], and poly-cyanoacrylate [PCA] (Pitt et al., 1981).
Nanoparticle delivery systems
Nanocapsules are vesicular systems in which a drug is confined toa cavity surrounded by a polymer membrane, whereas nanospheresare matrix systems in which the drug is physically and uniformlydispersed. Nanoparticles are solid, colloidal particles consisting ofmacromolecular substances that vary in size from 10 nm to 1000 nm(Kreuter, 1994a). However, particles N200 nm are not heavilypursued and nanomedicine often refers to devices b200 nm (i.e.,the width of microcapillaries). Typically, the drug of interest isdissolved, entrapped, adsorbed, attached and/or encapsulated intoor onto a nano-matrix. Depending on the method of preparationnanoparticles, nanospheres, or nanocapsules can be constructed topossess different properties and release characteristics for the bestdelivery or encapsulation of the therapeutic agent (Barratt, 2000;Couvreur et al., 1995; Pitt et al., 1981).
Applications and advantages of nanoparticle drug carriers
Polymeric nanoparticles made from natural and synthetic poly-mers have received the majority of attention due to their stability andease of surface modification (Herrero-Vanrell et al., 2005; Vauthier etal., 2003). They can be tailor-made to achieve both controlled drugrelease and disease-specific localization by tuning the polymercharacteristics and surface chemistry (Kreuter, 1994b; Moghimi etal., 2001; Panyam and Labhasetwar, 2003; Panyam et al., 2003b). It hasbeen established that nanocarriers can become concentrated prefer-
entially to tumors, inflammatory sites, and at antigen sampling sites byvirtue of the enhanced permeability and retention (EPR) effect of thevasculature. Once accumulated at the target site, hydrophobicbiodegradable polymeric nanoparticles can act as a local drug depotdepending on the make-up of the carrier, providing a source for acontinuous supply of encapsulated therapeutic compound(s) at thedisease site, e.g., solid tumors.
These systems in general can be used to provide targeted (cellularor tissue) delivery of drugs, improve bioavailability, sustain release ofdrugs or solubilize drugs for systemic delivery. This process can beadapted to protect therapeutic agents against enzymatic degradation(i.e., nucleases and proteases) (Ge, 2002). Thus, the advantages ofusing nanoparticles for drug delivery are a result of two main basicproperties: small size and use of biodegradable materials. Nanopar-ticles, because of their small size, can extravasate through theendothelium in inflammatory sites, epithelium (e.g., intestinal tractand liver), tumors, or penetrate microcapillaries. In general, thenanosize of these particles allows for efficient uptake by a variety ofcell types and selective drug accumulation at target sites (Desai et al.,1997; Panyam and Labhasetwar, 2003; Panyam et al., 2003b). Manystudies have demonstrated that nanoparticles have a number ofadvantages over microparticles (N1 μm) as a drug delivery system(Linhardt, 1989). Nanoparticles have another advantage over largermicroparticles because they are better suited for intravenous delivery.The smallest capillaries in the body are 5–6 μm in diameter. The size ofparticles being distributed into the bloodstream must be significantlysmaller than 5 μm, without forming aggregates, to ensure that theparticles do not cause an embolism.
The use of biodegradable materials for nanoparticle preparationallows for sustained drug releasewithin the target site over a period ofdays or even weeks. Biodegradable nanoparticles formulated fromPLGA and PLA have been developed for sustained drug delivery andare especially effective for drugs with an intracellular target (Barreraet al., 1993; Davda and Labhasetwar, 2002; Panyam and Labhasetwar,2003). Rapid escape of hydrophobic PCL-coated nanoparticles fromendo-lysosomes to the cytoplasm has been demonstrated (Barrera etal., 1993; Woodward et al., 1985). Greater and sustained anti-proliferative activity was observed in vascular smooth muscle cellsthat were treatedwith dexamethasone-loaded nanoparticles and thencompared to cells given drug in solution (Redhead et al., 2001). Hence,nanoparticles can be effective in delivering their contents tointracellular targets.
Characteristics important for drug delivery using nanoparticles
Particle size
Currently, the fastest and most routine method of determiningnanoparticle size is by photon-correlation spectroscopy or dynamiclight scattering. Photon-correlation spectroscopy requires the viscos-ity of the medium to be known and determines the diameter of theparticle by Brownian motion and light scattering properties (Swar-brick and Boylan, 2002). The results obtained by photon-correlationspectroscopy are usually verified by scanning or transmission electronmicroscopy (SEM or TEM).
Particle size and size distribution are the most importantcharacteristics of nanoparticles. They determine the in vivo distribu-tion, biological fate, toxicity, and targeting ability of these deliverysystems. In addition, they can influence drug loading, drug release,
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and stability of nanoparticles. Many studies have demonstrated thatnanoparticles have a number of advantages over microparticles(Panyam and Labhasetwar, 2003). Generally nanoparticles haverelatively high cell uptake when compared to microparticles and areavailable to a wider range of cellular and intracellular targets due totheir small size and mobility. Nanoparticles can cross the blood–brainbarrier (BBB) following the opening of endothelium tight junctions byhyper-osmotic mannitol, which may provide sustained delivery oftherapeutic agents for difficult-to-treat diseases like brain tumors(Kroll et al., 1998). Tween 80-coated nanoparticles have been shownto cross the BBB as well (Kreuter et al., 2003). Submicronnanoparticles, but not larger microparticles, are taken up by themajority of cell types (Zauner et al., 2001). Indeed, 100 nmnanoparticles had a 2.5-fold greater uptake rate than 1 μm micro-particles, and a 6-fold greater uptake than 10 μm microparticles byCaco-2 cells (Desai et al., 1997). In a similar study, nanoparticles wereshown to penetrate throughout the submucosal layers of a ratintestinal loop model, while microparticles were predominantlylocalized in the epithelial lining (Redhead et al., 2001). This indicatesthat particle distribution can, in part at least, be tuned by controllingparticle size.
Drug release is also affected by particle size. Smaller particles havea larger surface area-to-volume ratio; therefore, most of the drugassociated with small particles would be at or near the particlesurface, leading to faster drug release. In contrast, larger particles havelarge cores, which allow more drug to be encapsulated per particleand give slower release (Redhead et al., 2001). Thus, control of particlesize provides a means of tuning drug release rates.
Smaller particles also have a greater risk of aggregation duringstorage, transport, and dispersion. Polymer degradation also can beaffected by particle size. For instance, the rate of PLGA degradationwasfound to increase as the particle made from this polymer increased insize (Dunne et al., 2000). This process is believed to be due to PLGAdegradation products which can more easily diffuse through shorterdistances in smaller nanoparticles, while the polymer matrix of largerparticles increases the time of release due to the greater distance andmay also cause autocatalytic degradation of the polymer material(Panyam et al., 2003a). Therefore, it was hypothesized that largerparticles will contribute to faster polymer degradation as well as thedrug release. However, additional studies will be required to confirmand better elucidate the mechanisms of this paradox.
Surface properties of nanoparticles
The association of a drug to conventional carriers leads tomodification of the drug biodistribution profile, as it is mainlydelivered to the mononuclear phagocyte system (MPS) such as liver,spleen, lungs and bone marrow. Nanoparticles can be recognized bythe host immune system when intravenously administered andcleared by phagocytes from the circulation (Muller et al., 1996).Apart from the size of nanoparticles, nanoparticle hydrophobicitydetermines the level of blood components (e.g., opsonins) that bindthis surface. Hence, hydrophobicity influences the in vivo fate ofnanoparticles (Brigger et al., 2002; Muller et al., 1996). Indeed, once inthe blood stream, surface non-modified nanoparticles (conventionalnanoparticles) are rapidly opsonized and massively cleared by theMPS (Grislain et al., 1983).
To increase the likelihood of success in drug targeting, it isnecessary to minimize the opsonization and prolong the circulation ofnanoparticles in vivo. This can be achieved by coating nanoparticleswith hydrophilic polymers/surfactants or formulating nanoparticleswith biodegradable copolymers with hydrophilic characteristics, e.g.,polyethylene glycol (PEG), polyethylene oxide, polyoxamer, polox-amine, and polysorbate 80 (Tween 80). Studies show that PEG onnanoparticle surfaces prevents opsonization by complement andother serum factors. PEG molecules with brush-like and intermediate
configurations reduced phagocytosis and complement activation,whereas surfaces comprised of PEG with mushroom-like structureswere potent complement activators and favored phagocytosis (Bhadraet al., 2002; Olivier, 2005).
The zeta potential of a nanoparticle is commonly used tocharacterize the surface charge property of nanoparticles (Couvreuret al., 2002). It reflects the electrical potential of particles and isinfluenced by the composition of the particle and the medium inwhich it is dispersed. Nanoparticles with a zeta potential above±30 mV have been shown to be stable in suspension, as the surfacecharge prevents aggregation of the particles. The zeta potential alsocan be used to determine whether a charged active material isencapsulated within the center of the nanoparticle or on the surface.
Drug loading
A successful nanodelivery system should have a high drug-loadingcapacity, thereby reducing the quantity of matrix materials foradministration. Drug loading can be accomplished by two methods.The incorporation method requires the drug to be incorporated at thetime of nanoparticle formulation. The adsorption/absorptionmethodscall for absorption of the drug after nanoparticle formation; this isachieved by incubating the nano-carrier with a concentrated drugsolution. Drug loading and entrapment efficiency depend on drugsolubility in the excipient matrix material (solid polymer or liquiddispersion agent), which is related to the matrix composition,molecular weight, drug–polymer interactions, and the presence ofend functional groups (i.e., ester or carboxyl) in either the drug ormatrix (Govender et al., 1999; Govender et al., 2000; Panyam et al.,2004). A polymer of choice for some nanoparticle formulations isPEG, which has little or no effect on drug-loading and interactions(Peracchia et al., 1997). In addition, the macromolecules, drugs orprotein encapsulated in nanoparticles show the greatest loadingefficiency when they are loaded at or near their isoelectric point(pI) (Calvo et al., 1997). For small molecules, studies show the useof ionic interaction between the drug and matrix materials can bevery effective in increasing drug-loading (Chen et al., 1994, 2003).
Drug release
It is important to consider both drug release and polymerbiodegradation when developing a nanoparticulate delivery system.In general, the drug release rate depends on: (1) drug solubility; (2)desorption of the surface-bound or adsorbed drug; (3) drug diffusionthrough the nanoparticle matrix; (4) nanoparticle matrix erosion ordegradation; and (5) the combination of erosion and diffusionprocesses. Hence, solubility, diffusion, and biodegradation of theparticle matrix govern the release process.
In the case of nanospheres, where the drug is uniformlydistributed, drug release occurs by diffusion or erosion of the matrix.If the diffusion of the drug is faster than matrix erosion, then themechanism of release is largely controlled by a diffusion process. Therapid, initial release, or ‘burst’, is mainly attributed toweakly bound oradsorbed drug to the relatively large surface of nanoparticles(Magenheim et al., 1993). It is evident that the method of incorpora-tion has an effect on the release profile. If the drug is loaded by theincorporation method, then the system has a relatively small bursteffect and sustained release characteristics (Fresta et al., 1995). If thenanoparticle is coated by polymer, the release is then controlled bydiffusion of the drug from the polymeric membrane.
Membrane coating acts as a drug release barrier; therefore, drugsolubility and diffusion in or across the polymermembrane becomes adetermining factor in drug release. Furthermore, the release rate alsocan be affected by ionic interactions between the drug and auxiliaryingredients. When the entrapped drug interacts with auxiliaryingredients, a less water soluble complex can form, which can slow
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the drug release — having almost no burst release effect (Chen et al.,1994). Whereas if the addition of auxiliary ingredients, e.g., ethyleneoxide–propylene oxide block copolymer (PEO-PPO) to chitosan,reduces the interaction of the drug with the matrix material due tocompetitive electrostatic interaction of PEO-PPO with chitosan, thenan increase in drug release could be achieved (Calvo et al., 1997).Various methods can be used to study the release of drug from thenanoparticle: (1) side-by-side diffusion cells with artificial orbiological membranes; (2) dialysis bag diffusion; (3) reverse dialysisbag diffusion; (4) agitation followed by ultracentrifugation/centrifu-gation; or (5) ultra-filtration. Usually the release study is carried outby controlled agitation followed by centrifugation. Due to the time-consuming nature and technical difficulties encountered in theseparation of nanoparticles from release media, the dialysis techniqueis generally preferred. However, these methods prove difficult toreplicate and scale-up for industrial use.
Targeted drug delivery
The development of nanoparticle delivery systems for targeteddrug delivery has been recently reviewed (Moghimi et al., 2001).Targeted delivery can be actively or passively achieved. Activetargeting requires the therapeutic agent to be achieved by conjugatingthe therapeutic agent or carrier system to a tissue or cell-specificligand (Lamprecht et al., 2001). Passive targeting is achieved byincorporating the therapeutic agent into a macromolecule ornanoparticle that passively reaches the target organ. Drugs encapsu-lated in nanoparticles or drugs coupled to macromolecules canpassively target tumors through the EPR effect. Alternatively, catheterscan be used to infuse nanoparticles to the target organ or tissues. Forexample, localized delivery of drug-bearing nanoparticles to sites ofvascular restenosis may be useful for providing sustained drug releaseat specific sites on the arterial wall (Maeda, 2001; Sahoo et al., 2002).
Liposomes have been demonstrated to be useful for deliveringpharmaceutical agents. These systems use ‘contact-facilitated drugdelivery’, which involves binding or interaction with the targeted cellmembrane. This permits enhanced lipid–lipid exchange with the lipidmonolayer of the nanoparticle, which accelerates the convective fluxof lipophilic drugs (e.g., paclitaxel) to dissolve through the outer lipidmembrane of the nanoparticles to targeted cells (Guzman et al., 1996).Such nanosystems can serve as drug depots exhibiting prolongedrelease kinetics and long persistence at the target site.
Nanoparticles also can be formulated to deliver drugs acrossseveral biological barriers (Fisher and Ho, 2002; Lockman et al., 2002).Anti-neoplastics, anti-viral drugs, and several other types of drugs aremarkedly hindered because of inability of these molecules to cross theBBB. The application of nanoparticles to deliver across this barrier isextremely promising. It has been reported that nanoparticles can crossthe BBB following the opening of tight junctions by hyper-osmoticmannitol, which also may provide sustained delivery of therapeuticagents for difficult-to-treat diseases like brain tumors (Avgoustakis etal., 2002). Tween 80-coated nanoparticles also have been shown tocross the BBB (Beletsi et al., 1999).
Nanotechnology-based drug delivery in cancer
Drug delivery in cancer is important for optimizing the effect ofdrugs and reducing toxic side effects. Several nanotechnologies,mostly based on nanoparticles, can facilitate drug delivery to tumors.
Hydrogels
Hydrogel-nanoparticles are based on proprietary technology thatuses hydrophobic polysaccharides for encapsulation and delivery ofdrug, therapeutic protein, or vaccine antigen. A novel system usingcholesterol pullulan shows great promise. In this regard, four cholesterol
molecules gather to form a self-aggregating hydrophobic core withpullulan outside. The resulting cholesterol nanoparticles stabilizeentrapped proteins by forming this hybrid complex. These particlesstimulate the immune systemand are readily takenupbydendritic cells.Alternatively, larger hydrogels can encapsulate and release monoclonalantibodies.
Curcumin, a substance found in the cooking spice turmeric, haslong been known to have anti-cancer properties. Nevertheless,widespread clinical application of this relatively efficacious agenthas been limited due to its poor solubility and minimal systemicbioavailability. This problem has been resolved by encapsulatingcurcumin in a polymeric nanoparticle, creating “nanocurcumin”(Bisht et al., 2007). Further, the mechanism of action of nanocurcuminon pancreatic cancer cells mirrors that of free curcumin, includinginduction of apoptosis, blockade of nuclear factor kappa B (NFκB)activation, and downregulation of pro-inflammatory cytokines (i.e.,IL-6, IL-8 and TNF-α). Nanocurcumin provides an opportunity toexpand the clinical repertoire of this efficacious agent by enablingsoluble dispersion. Future studies utilizing nanocurcumin are war-ranted in preclinical in vivo models of cancer and other diseases thatmight benefit from the effects of curcumin.
Micelles and liposomes
Block-copolymer micelles are spherical super-molecular assembliesof amphiphilic copolymer. The core of micelles can accommodatehydrophobic drugs, and the shell is a hydrophilic brush-like corona thatmakes themicellewater soluble, thereby allowing delivery of the poorlysoluble contents. Camptothecin (CPT) is a topoisomerase I inhibitor thatis effective against cancer, but clinical application of CPT is limited by itspoor solubility, instability, and toxicity. Biocompatible, targeted steri-cally stabilized micelles (SSM) have been used as nanocarriers for CPT(CPT-SSM). CPT solubilization in SSM is expensive yet reproducible andis attributed to avoidance of drug aggregate formation. Furthermore,SSM composed of PEGylated phospholipids are attractive nanocarriersfor CPT delivery because of their size (14 nm) and ability to extravasatethrough the leakymicrovasculature of tumors and inflamed tissues. Thispassive targeting results in high drug concentration in tumors andreduced drug toxicity to the normal tissues (Koo et al., 2006).
Stealth micelle formulations have stabilizing PEG coronas tominimize opsonization of the micelles and maximize serum half-life.Currently, SP1049C, NK911, and Genexol-PM have been approved forclinical use (Sutton et al., 2007). SP1049C is formulated as doxorubicin(DOX)-encapsulated pluronic micelles. NK911 is DOX-encapsulatedmicelles from a copolymer of PEG-DOX-conjugated poly(asparticacid), and Genexol-PM is a paclitaxel-encapsulated PEG-PLA micelleformulation. Polymermicelles have several advantages over other drugdelivery systems, including increased drug solubility, prolongedcirculation half-life, selective accumulation at tumor sites, and lowertoxicity. However, at the present time this technology lacks tumorspecificity and the ability to control the release of the entrappedagents. Indeed, the focus of nano-therapy has gradually shifted frompassive targeting systems (e.g., micelles) to active targeting.
Super paramagnetic iron oxide particles can be used in conjunctionwith magnetic resonance imaging (MRI) to localize the tumor as wellas for subsequent thermal ablation. This has been used, for example, totarget glioblastoma multiforme (GBM), a primary malignant tumor ofthe brainwith few effective therapeutic options. The primary difficultyin treating GBM lies in the difficulty of delivering drugs across the BBB.However, nanoscale liposomal iron oxide preparations were recentlyshown to improve passage across the BBB (Jain, 2007).
Nanomaterial formulation
Nanomaterials have been successfully manipulated to create anew drug-delivery system that can solve the problem of poor water
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solubility of most promising currently available anticancer drugs and,thereby, increase their effectiveness. The poorly soluble anticancerdrugs require the addition of solvents in order for them to be easilyabsorbed into cancer cells. Unfortunately, these solvents not onlydilute the potency of the drugs but create toxicity. Researchers fromthe University of California Los Angeles California NanosystemInstitute have devised a novel approach using silica-based nanopar-ticles to deliver the anticancer drug CPT and other water insolubledrugs to cancer cells (Lu et al., 2007). The method incorporates thehydrophobic anticancer drug CPT into the pores of fluorescentmesoporous silica nanoparticles and delivers the particles into avariety of human cancer cells to induce cell death. The results suggestthat the mesoporous silica nanoparticles might be used as a vehicle toovercome the insolubility of many anticancer drugs.
Nanosystems
Novel nanosystems can be pre-programmed to alter their structureand properties during the drug delivery process, allowing for moreeffective extra- and intra-cellular delivery of encapsulated drug(Wagner, 2007). This is achieved by the incorporation of molecularsensors that respond to physical or biological stimuli, includingchanges in pH, redox potential, or enzymes. Tumor-targetingprinciples include systemic passive targeting and active receptortargeting. Physical forces (e.g., electric or magnetic fields, ultrasound,hyperthermia, or light) may contribute to focusing and triggeringactivation of nano systems. Biological drugs delivered with pro-grammed nanosystems also include plasmid DNA, siRNA, and othertherapeutic nucleic acids.
Using a degradable, polyamine ester polymer, polybutanedioldiacrylate co amino pentanol (C32), a diptheria toxin suicide gene(DT-A) driven by a prostate-specific promoter was directly injectedinto normal prostate and prostate tumors in mice (Peng et al., 2007).This C32/DT-A system resulted in significant size reduction, apoptosisin 50% of normal prostate. However, a single injection of C32/DT-Atriggered apoptosis in 80% of tumor cells present in the tissue. It isexpected that multiple nanoparticle injection would trigger a greatpercentage of prostate tumor cells to undergo apoptosis. These resultssuggest that local delivery of polymer/DT-A nanoparticles may haveapplication in the treatment of benign prostatic hypertrophy andprostate cancer.
Multidrug resistance (MDR) of tumor cells is known to developthrough a variety of molecular mechanisms. Glucosylceramidesynthase (GCS) is responsible for the activation of the pro-apoptoticmediator, ceramide, to a nonfunctional moiety, glucosylceramide. Thismolecule is over-expressed by many MDR tumor types and has beenimplicated in cell survival in the presence of chemotherapy. A studyhas investigated the therapeutic strategy of co-administering cer-amide with paclitaxel in an attempt to restore apoptotic signaling andovercome MDR in a human ovarian cancer cell line using modifiedpoly(epsilon-caprolactone) (PEO-PCL) nanoparticles to encapsulateand deliver the therapeutic agents for enhanced efficacy (van Vlerkenand Amiji, 2006). Results show that MDR cancer cells can becompletely eradicated by this approach. Using this approach, MDRcells can be resensitized to a dose of paclitaxel near the IC50 of non-MDR cells. Molecular analysis of activity verified the hypothesis thatthe efficacy of this therapeutic approach is due to a restoration inapoptotic signaling, showing the promising potential for clinical use ofthis therapeutic strategy to overcome MDR.
Nanocells
Indiscriminate drug distribution and severe toxicity of systemicadministration of chemotherapeutic agents can be overcome throughencapsulation and cancer cell targeting of chemotherapeutics in400 nm nanocells, which can be packaged with significant concentra-
tions of chemotherapeutics of different charge, hydrophobicity, andsolubility (MacDiarmid et al., 2007). Targeting of nanocells viabispecific antibodies to receptors on cancer cell membranes resultsin endocytosis, intracellular degradation, and drug release. Doses ofdrugs delivered via nanocells are ∼1000 times less than the dose ofthe free drug required for equivalent tumor regression. It producessignificant tumor growth inhibition and regression in mousexenografts and lymphoma in dogs, despite administration of minuteamounts of drug and antibody. Indeed, reduced dosage is a criticalfactor for limiting systemic toxicity. Clinical trials are planned fortesting this method of drug delivery.
Dendrimers
In early studies, dendrimer-based drug delivery systems focusedon encapsulating drugs. However, it was difficult to control the releaseof drugs associated with dendrimers. Recent developments inpolymer and dendrimer chemistry have provided a new class ofmolecules called dendronized polymers, which are linear polymersthat bear dendrons at each repeat unit. Their behavior differs fromthat of linear polymers and provides drug delivery advantages becauseof their enhanced circulation time. Another approach is to synthesizeor conjugate the drug to the dendrimers so that incorporating adegradable link can be further used to control the release of the drug.
DOXwas conjugated to a biodegradable dendrimer with optimizedblood circulation time through the careful design of size andmolecular architecture (Lee et al., 2006). Specifically, the DOX-dendrimer controlled drug-loading through multiple attachmentsites, solubility through PEGylation, and drug release through theuse of pH-sensitive hydrazone dendrimer linkages. In culture, DOX-dendrimers were N10 times less toxic than free DOX toward coloncarcinoma cells. Upon intravenous administration to tumor bearingmice, tumor uptake of DOX-dendrimers was nine-fold higher thanintravenous free DOX and caused complete tumor regression and100% survival of the mice after 60 days.
Nanotubes
Even though it was previously possible to attach drug moleculesdirectly to antibodies, attaching more than a handful of drugmolecules to an antibody significantly limits its targeting abilitybecause the chemical bonds that are used tend to impede antibodyactivity. A number of nanoparticles have been investigated toovercome this limitation. Tumor targeting single-walled carbonnano-tube (SWCNT) have been synthesized by covalently attachingmultiple copies of tumor-specific monoclonal antibodies (MAbs),radiation ion chelates and fluorescent probes (McDevitt et al., 2007). Anew class of anticancer compound was created that contains bothtumor-targeting antibodies and nanoparticles called fullerenes (C60).This delivery system can be loaded with several molecules of ananticancer drug, e.g., Taxol® (Ashcroft et al., 2006). It is possible toload as many as 40 fullerenes onto a single skin cancer antibody calledZME-108, which can be used to deliver drugs directly into melanomas.Certain binding sites on the antibody are hydrophobic (waterrepelling) and attract the hydrophobic fullerenes in large numbersso multiple drugs can be loaded into a single antibody in aspontaneousmanner. No covalent bonds are required, so the increasedpayload does not significantly change the targeting ability of theantibody. The real advantage of fullerene-based therapies vs. othertargeted therapeutic agents is likely to be fullerene's potential to carrymultiple drug payloads, such as taxol plus other chemotherapeuticdrugs. Cancer cells can become drug resistant, and one can cut downon the possibility of their escaping treatment by attacking them withmore than one kind of drug at a time. The first fullerene immuno-conjugates have been prepared and characterized as an initial steptoward the development of fullerene immunotherapy.
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Polymersomes
Polymersomes, hollow shell nanoparticles, have unique propertiesthat allow delivery of distinct drugs. Loading, delivery and cytosolicuptake of drug mixtures from degradable polymersomes were shownto exploit the thick membrane of these block copolymer vesicles, theiraqueous lumen, and pH-triggered release within endolysosomes.Polymersomes break down in the acidic environments for targetedrelease of these drugs within tumor cell endosomes. While cellmembranes and liposomes are created from a double layer ofphospholipids, a polymersome is comprised of two layers of syntheticpolymers. The individual polymers are considerably larger thanindividual phospholipids but have many of the same chemicalfeatures.
Polymersomes have been used to encapsulate paclitaxel and DOX forpassive delivery to tumor-bearing mice (Ahmed et al., 2006). The largepolymers making up the polymersome allows paclitaxel, which is waterinsoluble, to embed within the shell. DOX is water-soluble and stayswithin the interior of the polymersome until it degrades. The polymer-some and drug combination spontaneously self-assembles when mixedtogether. Recently, studies have shown that cocktails of paclitaxel andDOX lead to better tumor regression that either drug alone, butpreviously there was no carrier system that could carry both drugs asefficiently to a tumor. Hence, this approach shows great promise.
Quantum dots
Single-particle quantum dots conjugated to tumor-targeting anti-human epidermal growth factor receptor 2 (HER2) MAb have beenused to locate tumors using high-speed confocal microscopy (Tada etal., 2007). Following injection of quantum dot-MAb conjugate, sixdistinct stop-and-go steps were identified in the process as theparticles traveled from the injection site to the tumor where theybound HER2. These blood-borne conjugates extravasated into thetumor, bound HER2 on cell membranes, entered the tumor cells andmigrated to the perinuclear region. The image analysis of the deliveryprocesses of single particles in vivo provided valuable information onMAb-conjugated therapeutic particles, which will be useful inincreasing their anticancer therapeutic efficacy. However, the ther-apeutic utility of quantum dots remains undetermined.
Fig. 1. XPclad® nanoparticle formulation. The milling jar holds heat-absorbent zirconium oxiaround a common axis of the chamber wheel. This produces the rotation of planetary ballsstarch, polyethylene glycol, Texas red, and/or drug. By controlling the centrifugal force, varyzirconium planetary balls, duration and number of cycles, the size of the core can be controimpact and frictional forces causes the planetary balls to mill the contents in the jar.
XPclad® nanoparticles
The poor aqueous solubility of many drug candidates presentsa significant problem in drug delivery and related requirements suchas bioavailability and absorption. Recently, our laboratory hasdeveloped XPclad® nanoparticles that represent a novel formulationmethod that uses planetary ball milling to generate particles ofuniform size (Fig. 1), 100% loading efficiency of hydrophobic orhydrophilic drugs, subsequent coating for targeted delivery, andcontrol of LogP for systemic, cutaneous, or oral administration ofcancer drugs, vaccines, or therapeutic proteins (Fig. 2).
The method for making XPclad® nanoparticles uses a novel andrelatively inexpensive preparation technique (i.e., planetary ballmilling), which allows for controlling the size of the particles(100 nm to 50 μm; ±10% of mean size) with N99% loading efficiency,polymer- or ligand-coating for controlled-, protected-, and targeted-release and delivery of their contents. The nanoparticles producedthereby contain the desired biologically active agent(s) in abiopolymer excipient such as alginate, cellulose, starch or collagenand biologically active agents. Generally, there are two types of millsthat have been employed for making particles: vibratory or planetaryball mills. The vibratory ball milling grinds powders by high velocityimpact while planetary ball milling employs a grinding motion.Typically, planetary ball milling has been used only to generatemicron-sized particles, while vibratory milling can yield nano-particles. However, the high impact resulting from the vibratorymilling technique makes incorporating biologicals difficult. Planetaryball mills pulverize and mix materials ranging from soft and mediumto extremely hard, brittle and fibrous materials. Both wet and drygrinding can be carried out. Minerals, ores, alloys, chemicals, glass,ceramics, plant materials, soil samples, sewage sludge, householdand industrial waste and many other substances can be reduced insize simply, quickly and without loss. Planetary ball mills have beensuccessfully used in many industrial and research sectors, particu-larly wherever there is high demand for purity, speed, fineness andreproducibility. The planetary ball mills produce extremely highcentrifugal forces with very high pulverization energies and shortgrinding times. Because of the extreme forces exerted, the useof vibratory and planetary ball mills to formulate therapeuticshas not been practiced until now. In general, XPclad® particle size
de planetary milling balls, rotates about its own axis as well as in the opposite direction,and enables the milling of particles from macroparticles containing materials such asing the revolutions/s of Ω, jar velocity (ω), radius (R), the size as well as number of thelled to generate 5–30 nm up to 10–60 μm particles. In this system, the combination of
Fig. 2. Overview of XPclad® nanoparticle applications. (Panel A) XPclad® nanoparticles produced by planetary ball milling contain the desired biologically active agent(s) in abiopolymer excipient such as alginate, cellulose, starch or collagen and biologically active agents with N99% loading efficiency. XPclad particles are coated with polymers and/orligands for controlled, protected, and targeted delivery of their contents. (Panel B) XPclad® particle size can be engineered to various size ranges. (Panel C) Mice receiving dendriticcell-binding peptide-coated XPclad® nanoparticles containing Streptococcus pneumonia pneumococcal surface protein A (PspA) peptide plus TLR7/8 agonist as adjuvant caused asignificant reduction in viable bacteria after challenge compared to similarly challenged naïve animals or control mice. (Panel D) XPclad® nanoparticles selectively target prostatecancer (PC3) cells but do not kill normal prostate epithelial cells (RWPE-1). Similarly, PC3 tumor-bearing mice that received folic acid-coated XPclad® nanoparticles containingcisplatin showed significant tumor regression compared to similar control mice. (Panel E) Passive immunity to anthrax toxins was effected by oral delivery of anti-protective antigen(PA) MAbs using XPclad® nanoparticles to neutralize anthrax toxin after systemic, oral, or respiratory exposure.
221R. Singh, J.W. Lillard Jr. / Experimental and Molecular Pathology 86 (2009) 215–223
can be engineered to range from 5 to 30 nm up to 10 to 60 μm bycontrolling the size and number of planetary balls, grinding speed,milling cycles, and centrifugal force by varying the revolutions persecond and planetary jar velocity.
The surface of XPclad® nanoparticles can be modified withhydrophilic (e.g., PEG) and/or hydrophobic (e.g., PCL) polymers toprecisely control LogP values. Surface polymers can be modifiedthrough the conjugation of targeting molecules (e.g., antibodies,folate, etc.) to active delivery of encapsulated agents. The interiorcore can entrap hydrophobic or hydrophilic molecules (e.g., drug,immune adjuvant, nucleic acid, metal ion, fluorophore, therapeuticprotein, and/or peptide). For example, PC3 tumor-bearing micethat received folic acid-coated XPclad® nanoparticles containingTexas red plus cisplatin showed significant tumor regressioncompared to similar control mice (Fig. 2). Moreover, XPclad®
nanoparticles selectively induced PC3 cell death but did not kill normalepithelial cells of similar origin (RWPE-1 cells). Mice receiving dendriticcell-binding peptide-coated XPclad® nanoparticles containing Strepto-coccus pneumonia pneumococcal surface protein A (PspA) peptide andTLR7/8 agonist as adjuvant showed significant reduction in bacterialload after challenge compared to similarly challenged naïve animalsor control mice that did not receive nanovaccines with PspA peptideor toll-like receptor (TLR)7/8 adjuvant. Therapeutic proteins (e.g.,antibody) also can be encapsulated in XPclad® particles (4–12 μm)for oral delivery. Passive immunity can also be afforded by oraldelivery of anti-protective antigen MAbs using XPclad® nanoparti-
cles to neutralize anthrax toxin after systemic, oral, or respiratoryexposure.
Conclusions
Nano delivery systems hold great potential to overcome some ofthe obstacles to efficiently target a number of diverse cell types. Thisrepresents an exciting possibility to overcome problems of drugresistance in target cells and to facilitate the movement of drugsacross barriers (e.g., BBB). The challenge, however, remains theprecise characterization of molecular targets and ensuring that thesemolecules only affect targeted organs. Furthermore, it is important tounderstand the fate of the drugs once delivered to the nucleus andother sensitive cell organelles.
Disclosure of potential conflicts of interest
The authors of this manuscript have a patent pending (PCT/US2007/006844) for the manufacture and use of XPclad® particles fordelivering drugs, proteins, peptides, and nucleic acids.
Acknowledgments
The content of this manuscript benefited frommany fruitful conversa-tions with members of the Morehouse School of Medicine and theUniversityof Louisville. Thisworkbenefited fromthe cooperationbetween
222 R. Singh, J.W. Lillard Jr. / Experimental and Molecular Pathology 86 (2009) 215–223
investigators from the Morehouse School of Medicine and the WallaceTumor Institute at the University of Alabama at Birmingham via theNational Cancer Institute sponsored “ComprehensiveMinority Institution/Cancer Center Partnership”. This review and the studies described hereinwere supported in part by funds from the Smith and Lucille GibsonEndowment, Department of Defense Prostate Cancer Research Programawards and National Institute of Health Grants AI057808, CA092078,CA086359, DK58967, GM08248, GM09248, MD00525, and RR03034.
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