Pharmaceutical Research, Vol. 13, No. 2, 1996
`
`Report
`
`Formulation and Antitumor Activity
`Evaluation of Nanocrystalline
`Suspensions of Poorly Soluble
`Anticancer Drugs
`
`E. Merisko-Liversidge,1.3 P. Sarpotdar,1 J. Bruno,1
`S. Haij,1 L. Wei,1 N. Peltier,1 J. Rake,1 J.M.
`Shaw,1 S. Pugh,2 L. Polin,2 J. Jones,2 T. Corbett,2
`E. Cooper,1 and G. G. Liversidge1
`
`Purpose. Determine if wet milling technology could be used to
`formulate water insoluble antitumor agents as stabilized nanocrystalline
`drug suspensions
`that retain biological effectiveness following
`intravenous injection.
`Methods. The versatility of the approach is demonstrated by evaluation
`of four poorly water soluble chemotherapeutic agents that exhibit
`diverse chemistries and mechanisms of action. The compounds selected
`were: piposulfan (alkylating agent), etoposide (topoisomerase II inhibi(cid:173)
`tor), camptothecin (topoisomerase I inhibitor) and paclitaxel (antimi(cid:173)
`totic agent). The agents were wet milled as a 2% w/v solids suspension
`containing I% w/v surfactant stabilizer using a low energy ball mill.
`The size , physical stability and efficacy of the nanocrystalline suspen(cid:173)
`sions were evaluated.
`Results. The data show the feasibility of formulating poorly water
`soluble anticancer agents as physically stable aqueous nanocrystalline
`suspensions. The suspensions are physically stable and efficacious
`following intravenous injection.
`Conclusions. Wet milling technology is a feasible approach for formu(cid:173)
`lating poorly water soluble chemotherapeutic agents that may offer a
`number of advantages over a more classical approach.
`KEY WORDS: anticancer agents; poorly water soluble agents; nano(cid:173)
`particles; etoposide; camptothecin; piposulfan and paclitaxel.
`
`INTRODUCTION
`
`Frequently in drug discovery poorly water soluble agents
`are identified as actives using in vitro assays but are discarded
`because they are unable to be formulated for further evaluation
`in vivo. Thus many promising agents are discarded due to
`poor water solubility and the lack of a generally applicable
`formulation approach to address this problem. Though this
`predicament is a likely scenario in any therapeutic/diagnostic
`discovery program it has been a recurring issue in cancer
`research. The development of a number of promising anticancer
`agents have been delayed or abandoned due to issues resulting
`from the poor water solubility of the drug (l).
`Currently, the poorly water soluble chemotherapeutic
`agents used in the clinic and that are in various phases of
`development are formulated by conventional methods. Rou(cid:173)
`tinely, these agents are formulated using a co-solvent plus other
`excipients which act to solubilize the drug and provide for
`stability of the formulation during storage and on injection (2).
`
`1 NanoSystems, Collegeville, Pennsylvania 19426.
`2 Division of Hematology and Oncology, Wayne State University,
`Detroit, Michigan 48202.
`3 To whom correspondence should be addressed.
`
`Commonly, the use of co-solvents produce toxic side effects
`e.g., anaphylaxis, pain at the site of injection, emboli formation
`and paradoxically precipitation which results in poor intrave(cid:173)
`nous bioavailability. For instance, etoposide (VP-16), a semi(cid:173)
`synthetic analog of podophy llotoxin, is a sparingly water soluble
`drug that is used to treat small-cell lung cancer and various
`other neoplasms (3,4). Etoposide being a poorly water soluble
`drug is formulated using benzyl alcohol, polyoxyethylated sor(cid:173)
`bitan ester (Tween 80) and polyethyleneglycol (PEG 300). Prior
`to injection the formulation is diluted then slowly infused. Even
`though etoposide has been used in the clinic for a number of
`years, formulation related toxicity issues still occur (5-7).
`Extensive literature is available on other technologies con(cid:173)
`cerned with delivery issues of hard to formulate chemotherapeu(cid:173)
`tic agents. Although significant progress has been made in each
`of these areas including liposomes (8,9), emulsions (10,11)
`and polymeric carriers (12,13), there is a need for additional
`methodologies that provide a safe, effective and economical
`means for intravenous administration of poorly water soluble
`therapeutics and/or diagnostics.
`In this study, a delivery system well suited for sparingly
`water soluble chemotherapeutic agents is described wherein the
`compounds are formulated as dispersible particles consisting
`essentially of a crystalline drug substance stabilized with a
`surface modifier. The methods of preparation and characteriza(cid:173)
`tion of nanocrystalline dosage forms suitable for intravenous
`administration of poorly water soluble chemotherapeutic agents
`are presented together with preclinical efficacy data performed
`in the mammary 16C murine tumor model.
`
`MATERIALS AND METHODS
`
`Chemicals
`
`With the exception of piposulfan which was custom syn(cid:173)
`thesized at Kodak Research Laboratories, Rochester, NY.,
`agents were obtained from the following vendors: etoposide
`(Sigma Chemical Co, St. Louis, MO), camptothecin (Aldrich,
`Milwaukee, WI) and paclitaxel (Biolyse Corporation, Port-Dan(cid:173)
`iel, Que.). Pluronic and Tetronic surfactant stabilizers were
`obtained from BASF (Parsnippany, NJ.) and the polyoxyethy(cid:173)
`lated sorbitan esters were purchased from ICI, Wilmington, DE.
`
`Nanoparticle Formulations
`
`Nanoparticle formulations were prepared under aseptic
`conditions as a drug suspension (2% wt/v) containing 1 % (wt/v)
`surfactant stabilizer ( 14, 15). An aqueous suspension containing
`drug and stabilizer was wet milled using preconditioned zirco(cid:173)
`nium oxide media (Zircoa Inc., Solon, OH.) For screening,
`milling was performed in a 28 ml bottle using a media bead
`volume of 7.5 ml and 3.75 ml of the drug/surfactant slurry.
`The slurry was milled on a low energy mill (U.S. Stoneware,
`East Palestine, OH) at 57% of the critical speed. The critical
`speed is defined as the rotational speed of the grinding vessel
`when centrifuging of the grinding media occurs. Milling effi(cid:173)
`ciency was dependent on a number of factors including drug
`substance and choice of stabilizer. The particle size of the slurry
`was assayed daily during milling. Routinely, the drug/surfactant
`slurry was milled to a final size of less than 400 nm based on
`
`272
`
`

`

`Nanosuspensions of Anticancer Drugs
`
`273
`
`photon correlation spectroscopy (PCS) and generally this could
`be achieved in a 4 day period. Milled nanosuspensions were
`evaluated for chemical stability, physical stability, and physical
`stability in plasma. Formulations with acceptable physical sta(cid:173)
`bility in plasma were submitted for efficacy studies. Acceptable
`physical stability was defined as absence of agglomeration or
`negligible particle size growth in the presence of plasma when
`the nanosuspension was incubated with plasma for 60 min
`at 37°C.
`The stabilizers used for each of the formulations were:
`1) polyoxyethylene sorbitan monooleate (Tween 80)1sorbitan
`monooleate (Span 80) for piposulfan; 2) Pluronic F108 for
`camptothecin, and 3) Pluronic F127 for etoposide and paclitaxel
`(Table 1).
`
`Particle Size Analysis
`
`Particle size analysis was determined using photon correla(cid:173)
`tion spectroscopy (PCS). Prior to sizing, samples were diluted
`with freshly filtered deionized water. Sizing measurements were
`routinely performed using the Coulter Model N4MD Submicron
`Particle Analyzer (Coulter, Miami, Fl.) or the Malvern Zetamas(cid:173)
`ter (Malvern Instruments Ltd., Worcester, England) using uni(cid:173)
`modal analysis of intensity distributions for mean particle size·
`determination. PCS results were confirmed using scanning elec(cid:173)
`tron microscopy (SEM). For SEM, samples were diluted and
`an aliquot of the diluted preparation was dried, sputter coated
`and visualized using the Topco SM510 (Topcon Technologies,
`Inc., Pleasanton, Ca.).
`
`Plasma Stability Assays
`
`Stability of nanoparticle suspensions in plasma was moni(cid:173)
`tored by photon correlation spectroscopy (PCS) and light
`microscopy. Rat plasma was delipidated prior to use by centrifu(cid:173)
`gation at 150,000 X g for lhr. The supernatant was carefully
`decanted and filtered through a 0.1 micron filter. The plasma
`was then pretested in the Coulter N4MD submicron particle
`analyzer to ensure that the sample was particle-free. Nanosus~
`pensions of piposulfan, camptothecin, etoposide and paclitaxel
`were diluted 1:2, 1:10, and 1:100 with lipid-free plasma. Sam(cid:173)
`ples were vortexed and incubated at 37°C. For PCS analysis,
`samples were diluted with water and assayed. Presence or
`absence of aggregation was also monitored using the Leica
`DMRB optical microscope with a 100 X phase-contrast
`objective.
`
`Animal Studies
`Studies were performed in accordance with Wayne State
`University Medical School's animal use and care administrative
`
`policies. As previously described (16), tumor fragments (-30
`mg) were seeded subcutaneously by trocar into 7-10 week-old
`female C3H mice (National Institute of Health, Bethesda, Md.).
`Chemotherapy of either a nanoparticle suspension or a control
`formulation was initiated within 6 days of tumor implantation.
`For chemotherapy, animals were injected via the tail vein using
`a multiple injection regimen. Dose and dosing schedule were
`selected based on the toxicity of the compound with the inten(cid:173)
`tion of administering the drug at its maximum tolerated dose
`(MTD).
`To assess antitumor effectiveness, median tumor burden
`of treated animals was compared to median tumor burden of
`untreated controls. Results are expressed as a percentage deter(cid:173)
`mined by comparing the average weight of tumors in treated
`animals (expressed as "T") to the average weight of tumors in
`untreated controls (expressed as "C"). This value is expressed
`as percent TIC. A TIC = 0% is indicative of a highly active
`agent while a TIC >42% is considered inactive (16).
`
`RESULTS
`
`Particle Size Reduction
`
`Figure 1 shows the particle size reduction profile achieved
`during ball milling. Generally, within the first 24 h a significant
`reduction in particle size is observed. Depending on the drug
`core and stabilizer utilized, additional milling rendered finer
`particle dispersions. For this study, nanosuspensions were
`milled for 4 days and particle size analysis was performed every
`12 hrs. After four days of milling nanosuspensions of piposulfan
`and etoposide with a mean diameter -200 nm were harvested.
`For camptothecin and paclitaxel, milling was continued for an
`additional 3 to 4 days to further reduce particle size. As shown
`in Table 1, routinely nanosuspensions were prepared with a
`mean particle size that averaged 200-250 nm for all four
`agents studied.
`
`Characterization of Nanoparticle Drug Suspensions
`
`In Figure 2, the particle size distribution for nano-piposul(cid:173)
`fan, nano-etoposide, nano-camptothecin and nano-paclitaxel is
`shown. Intensity distributions were obtained using photon corre(cid:173)
`lation spectroscopy (PCS). Different drug cores with an optimal
`stabilizer produce a relatively uniform dispersion. The gross
`uniformity or homogeneity of the dispersions was verified using
`scanning electron microscopy (SEM). In Figure 3, the scanning
`electron micrographs show representative images of the nano(cid:173)
`particle drug suspensions. The milled nanosuspensions for each
`agent were homogeneous. However, the morphology of the
`
`Table 1. Average Particle Size: Nano-Suspensions•
`
`Compound (solubility)h
`
`Piposulfan (125µg/ml)
`Camptothecin (50µg/ml)
`Etoposide (200µg/ml)
`Paclitaxel (< 10 µg/ml)
`
`Formulation
`
`Average Size in nm. (number of batches)
`
`2% Piposulfan, 0.33% Tween 80, 0.67% Span 80
`2% Camptothecin, 1% F108
`2% Etoposide, 1% F127
`2% Paclitaxel, 1 % F127
`
`210.2 ± 38.9 (8)
`202.3 ± 30.5 (6)
`256.2 ± 53.0 (12)
`279.2 ± 29.60 (7)
`
`• Chemotherapeutic agents were roller milled as a 2% wt/v suspension using surfactant satbilizers at a core to surfactant ratio of 2: 1. Samples
`were sized using photon correlation spectroscopy (PCS). The average size was determined using the said number of batches.
`b Aqueous solubility @ 25°C.
`
`

`

`274
`
`Merisko-Liversidge et al.
`
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`Fig. 1. Effectiveness of particle size reduction using wet milling tech(cid:173)
`nology to formulate water insoluble chemotherapeutic agents. Com(cid:173)
`(piposulfan-D; · camptothecin-+ ; etoposide__.; and
`pounds
`paclitaxel-0) were roller milled using the surfactant stabilizers
`described in Table 1. During milling, particle size was monitored every
`12 hrs. For particle size analysis samples were diluted with deionized
`water, vortexed and sized using photon correlation spectroscopy (PCS).
`Data show weight average diameters of particles in suspension.
`
`various nanosuspensions differed from the cuboidal appearance
`of nanocamptothecin (Fig. 3B) to the rather elongated rod-like
`structures of nano-paclitaxel (Fig. 3D). Also, as monitored by
`both PCS and SEM, heterogeneity with respects to particle
`size and shape appears to be primarily governed by interactive
`properties of both the drug core and the surfactant stabilizer
`used during milling. When optimally stabilized, the suspensions
`did not aggregate and remained physically stable for at least
`four weeks post preparation (Fig. 4).
`Since the intended use of these nanosuspensions is for
`intravenous administration, physical stability in the presence
`of plasma was monitored using particle size analysis and optical
`microscopy. Nanosuspensions were diluted with plasma and
`incubated at 37°C . As shown in Figure 5, for camptothecin
`and paclitaxel no significant change could be detected in the
`mean particle size of the preparations throughout a 60 min
`incubation period. For piposulfan and etoposide, particle growth
`was observed but there was no evidence of particle aggregation
`and/or agglomeration based on both PCS sizing and optical
`
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`
`Particle Diameter (nm)
`Fig. 2. Size distribution profiles of nanoparticle suspensions were
`obtained using the Malvern Submicron Particle Sizer. Samples were
`diluted with deionized water, vortexed for -20 sec and sized. Size
`distribution profiles and cumulant z averages are shown for: • nanopi(cid:173)
`posulfan (z Ave = 299.9); 0 nanocamptothecin (z Ave = 227.8; o
`nanoetoposide (z Ave = 155.4); and• nanopaclitaxel (z Ave = 291.3).
`
`microscopy. Physical stability of the nanocrystalline suspen(cid:173)
`sions was also not affected by time of incubation and dilution.
`Samples diluted 1:100 with plasma and incubated at 37°C for
`24 hrs remained physically stable.
`
`Efficacy of Nanoparticle Drug Suspensions
`
`To evaluate efficacy, the nanocrystalline drug suspensions
`were tested in mice previously injected with mammary adeno(cid:173)
`carcinoma (16/C). The nanosuspensions were compared with
`the conventional formulation of the same agent or an agent
`of similar mechanism of action. Results of these studies are
`illustrated in Table 2. For all nanoparticle suspensions evaluated,
`tumor regression expressed as the percentage of tumor weight
`in treated animals to tumor weight in untreated controls was
`significant. In addition, the suspensions were suitable for intra(cid:173)
`venous bolus injection as suggested without increased incidence
`of acute toxicity in comparison to controls. For certain drugs,
`such as piposulfan, only the nanoparticle formulation was toler(cid:173)
`ated as an i.v. injectable. Control formulations were extremely
`toxic unless administered subcutaneously.
`
`DISCUSSION
`
`A technology is described for formulating water insoluble
`anticancer agents as nanoparticle drug suspensions that are
`stable. The method is versatile and suitable for many agents
`. whose solubility is less than 200 ug/ml. The potential value of
`this approach was demonstrated using two well-known drugs,
`etoposide and paclitaxel, which are in the clinic but have report(cid:173)
`edly been associated with formulation-related safety issues (6-
`8,17-19). The other agents
`investigated, piposulfan and
`camptothecin, are older drug candidates that could not be suc(cid:173)
`cessfully formulated as an injectable. In the case of piposulfan
`
`

`

`Fig.· 3: Nanocrystalline drug suspensfons were analyzed using scanning electroll microscopy (SEM). Unmllled drug
`substance was visualized at 500 X magnification and is shown on the left side of the figure as: A) piposulfan; B)
`camptothecin; C) etoposide; and D) paclitaxel. The corresponding nanocrystalline suspensions (5,000 X) are shown on
`the right side of the figure labeled as: E) nanopiposulfan; F) nanocamptothecin; G) nanoetoposide; and H) nanopaclitaxel.
`
`

`

`l
`
`276
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`
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`
`Initial Size
`
`4 Weeks
`
`•
`
`Nanopiposulfan
`
`El Nanocamptothecin
`ISi Nanopaclitaxel
`Nanoetoposide
`•
`Fig. 4. Physical stability of nanocrystal suspensions were studied at
`ambient temperature for a four week period. The antitumor agents ·
`were roller milled as described in Table 1 and Figure 1. The suspensions
`were then stored at room temperature for four weeks and re-sized
`using PCS. The graph compares mean particle size of the preparations
`immediately after milling with the mean particle size of the suspension
`following storage at room temperature for one month.
`
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`Fig. 5. The physical stability of nanocrystal drug suspensions was
`studied in the presence of plasma. Nanopiposulfan-D, nanocampto(cid:173)
`thecin-+, nanoetoposide-11, and nanopaclitaxel-0 were diluted
`l :2 with plasma and incubated for 60 min at 37°C. For particle size
`analysis samples were diluted with deionized water and sized using
`PCS. PCS size analysis was confirmed using optical microscopy.
`
`Merisko-Liversidge et al.
`
`drug development was abandoned, whereas, solubility issues
`associated with the camptothecins are being aggressively pur(cid:173)
`sued via the identification of water soluble analogs (20-23).
`The solubility of the anticancer agents chosen for this study
`range from -200 ug/ml to less than 4.0 ug/ml (24,25) and as
`demonstrated in this study can be readily formulated as an
`aqueous suspension of fine particles using a low energy wet
`milling process .
`The nanoparticle suspensions of piposulfan, camptothecin,
`etoposide and paclitaxel that are described were generated using
`a conventional ball mill. As the data show, in the presence of
`the selected surfactant stabilizer(s) the procedure was effective
`in producing nanoparticles of pure drug substance. Generally,
`the higher molecular weight polymeric stabilizers were optimal
`for effective particle size reduction, shelf stability, and preven(cid:173)
`tion of agglomeration in the presence of blood proteins. For
`instance, stable suspensions -250 nm in diameter were obtained
`for camptothecin, etoposide and paclitaxel using the pluronic
`block co-polymers Fl 08 and Fl 27. The higher molecular weight
`Pluronics have been shown to be excellent stabilizers for various
`colloidal delivery systems (26). In addition, these surfactant
`coatings have been known to reduce opsonization of particulate
`drug carriers and enhance delivery of the desired agent to
`various anatomical targets which would be advantageous for
`passive delivery to solid tumors (27 ,28). The pharmacokinetic
`properties of these nanoparticle drug suspensions are being
`studied. However, since the technology described in this study
`is relatively new and the biodistributional properties of colloidal
`nanocarriers are dictated by a complexity of interactions, it
`would be surprising if the blood clearance of these nanosuspen(cid:173)
`sions is not rapid. Currently, methodology is being developed
`so that properties of the nanosuspensions, e.g. size, surface
`characteristics and shape can be modulated to optimize delivery.
`Though the effects of the comminution process on the
`physical state of the drug was not performed, previous studies
`using X-ray diffraction have shown that the dispersion process
`did not change the crystal structure of the compound (14). In
`this study the electron micrographs of the milled dispersions
`suggest that the process generates nanocrystalline drug particles.
`However, comparisons between pre and post processed materi(cid:173)
`als remains to be studied.
`For certain drugs such as piposulfan, generating a stable
`nanocrystalline suspension proved challenging. After screening
`a series of surfactants and surfactant combinations, the use of
`a Tween 80 and Span 80 mixture provided optimal physical
`stabilization. As shown in Figure 1 and 2, using this surfactant
`combination, particle size of the suspension was reduced to
`250 nm in -4 day period. For piposulfan the su1factant mixture
`adequately wets the drug substance and provides steric stabiliza(cid:173)
`tion. However, as judged from the plasma stability data shown
`in Figure 5, though the preparation does not agglomerate in
`plasma, the particles apparently interact with plasma proteins
`resulting in an overall increase in mean particle size of the
`preparation. This interaction does not appear to compromise
`the safety and efficacy of the suspension (Table 2).
`As shown in Table 2, the efficacy of oncologic agents when
`formulated as nanoparticles was satisfactory. For etoposide and
`paclitaxel, novel nanoparticle suspensions were compared to
`currently used clinical formulations. Etoposide is formulated
`using polyethylene glycol, Tween 80 and benzyl alcohol while
`paclitaxel is dissolved in a mixture of Cremophor EL and
`ethanol. To avoid acute toxicity, both formulations must be
`
`

`

`Nanosuspensions of Anticancer Drugs
`
`Table II. Efficacy of Nanoparticle Suspensions Against Early Stage Mammary Adenocarcinoma 16C in CH3 Mice•
`
`Formulation
`
`NanoPiposulfan
`Comparator I
`No Treatment
`NanoCamptothecin
`Comparator II
`
`No Treatment
`NanoEtoposide
`Comparator III
`
`No Treatment
`NanoPaclitaxel
`Comparator IV
`No Treatment
`
`Schedule
`
`BID 1-4
`BID 1-4
`NA
`d 1-3
`d 1-3
`d 1-3
`NA
`d 1,5
`d 1,3,5
`d 1,3,5
`d 1,3,5
`NA
`d 3.6
`BID 3,4,5
`NA
`
`Total Dose
`(mg/kg)
`
`Drug Deaths
`(#deaths/total)
`
`Median Tumor Burden
`(mg)
`
`356
`360
`NA
`40
`20
`12.5
`NA
`125
`102
`69
`45
`NA
`88
`100
`NA
`
`015
`2/5
`015
`015
`515
`1/5
`015
`015
`515
`4/5
`0115
`015
`015
`315
`015
`
`0 (0-234)
`32 (0-63)
`3582 (1352-4678)
`907 (284-2514)
`NA
`969 (352-1224)
`2576 (627-3054)
`0 (0-88)
`NA
`75 (75)
`171 (0-260)
`1387 (0-2016)
`53 90-196)
`158 (138-839)
`2674 (0-3043)
`
`277
`
`TIC
`%
`
`0
`1.0
`NA
`35
`NA
`38
`NA
`0
`NA
`5
`12
`NA
`3
`7
`NA
`
`• Formualtions were injected intravenously using the stated schedule and dosage. Comparator formulations were: a) Comparator I: cyclophosamide
`in saline; 2) Comparator II: camptothecin in 5% ethanol, 2% POE40; c) Comparator III: etoposide in 7% ethanol, 3% POE40 and d) Comparator
`IV: paclitaxel in 50% ethanol, 50% Cremophor. Once a day and twice daily injections are denoted by "d" and "BID" respectively.
`
`diluted prior to use and slowly infused. In the present study,
`these formulations were slowly injected, whereas, nanoparticu(cid:173)
`late suspensions were administered via a rapid bolus injection.
`The data show that in the mammary 16C murine tumor model,
`the performance of nano-etoposide and nano-paclitaxel was
`comparable or slightly improved over the response elicited by
`the conventional formulations. In addition, the data clearly show
`that when administered as a fine particle preparation, dosages
`could be increased without incidence of acute toxicity, abnormal
`weight loss, or organ pathology observed during necropsy. Since
`the surfactants used in the nanocrystal formulations were
`selected so as to prevent agglomeration in the presence of
`plasma, the improved acute safety profile of the suspension
`was anticipated. However, other factors such as an altered phar(cid:173)
`macokinetic profile should be considered.
`Our data suggest that the ability to formulate water insolu(cid:173)
`ble chemotherapeutic agents as nanocrystal drug suspensions
`may offer a number of advantages over a more classical
`approach. First, since these fine particle drug suspensions are
`well tolerated following intravenous injection, nanoparticles
`provide a convenient remedy for administering high doses of
`drug without the risks routinely associated with conventional
`formulations containing cosolvents. Second, the potential for
`being able to safely increase the dosage of a particular drug
`may lead to the identification of new medical indications and
`usages for the given drug. Lastly, the technology described in
`this study provides a convenient platform for targeted delivery
`of water insoluble therapeutic and/or diagnostic agents.
`
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