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Brain uptake of thiamine-coated nanoparticles
Journal of Controlled Release 93 (2003) 271 – 282 www.elsevier.com/locate/jconrel
Brain uptake of thiamine-coated nanoparticles Paul R. Lockman a, Moses O. Oyewumi b, Joanna M. Koziara b, Karen E. Roder a, Russell J. Mumper b, David D. Allen a,* a
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, 1300 So. Coulter Drive, Amarillo, TX 79106-1712, USA b Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA Received 15 May 2003; accepted 18 August 2003
Abstract Recently, a novel nanoparticle (NP) comprised of emulsifying wax and Brij 78 was shown to have significant brain uptake using the in-situ rat brain perfusion technique. To further these studies and to specifically target brain, we have incorporated thiamine as a surface ligand on the nanoparticles. Solid nanoparticles were prepared from oil-in-water microemulsion precursors. Nanoparticles were radiolabeled and a thiamine ligand (thiamine linked to distearoylphosphatidylethanolamine via a polyethylene glycol spacer) was coated on the surface of the nanoparticles. Initial experiments focused on assessing uptake of [3H]nanoparticles with and without thiamine surface ligands. Biodistribution nanoparticle studies were also carried out in BALB/c mice. The results showed: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of nanoparticles with and without the thiamine surface ligands, and (3) initial data suggesting mechanisms for nanoparticle brain entry. Comparison of NP brain uptake demonstrated that the thiamine-coated nanoparticle associated with the blood – brain barrier (BBB) thiamine transporter and had an increased Kin between 45 and 120 s (thiamine coated NP 9.8 F 1.1 10 3 ml/s/g versus uncoated NPs; 7.0 F 0.3 10 3 ml/s/g). It was concluded that the thiamine ligand facilitated binding and/or association with blood – brain barrier thiamine transporters, which may be a viable mechanism for nanoparticle mediated brain drug delivery. D 2003 Elsevier B.V. All rights reserved. Keywords: Microemulsions; Blood – brain barrier; Emulsifying wax; Targeting; Biodistribution
1. Introduction Effective brain drug delivery is limited by the blood – brain barrier (BBB). This interface between plasma and brain consists of both brain microvascular endothelium and the surrounding glia. The BBB * Corresponding author. Tel.: +1-806-356-4000x286; fax: +1806-356-4034. E-mail address: [email protected] (D.D. Allen). 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.08.006
significantly restricts water-soluble, charged and high molecular weight therapeutics to the vascular space while allowing brain parenchyma penetration of small and/or lipophilic molecules [1]. Mechanisms of permeability regulation include: (1) microvascular endothelial tight junctions [2], (2) enzymatic regulation [3], and (3) active brain efflux [4]. While multiple strategies have been employed to circumvent the BBB, an emerging approach is the use of nanoparticles (NPs). The composition of the nanoparticles can be adjusted
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to allow almost any therapeutic drug to be adsorbed or entrapped. This method effectively disguises the membrane barrier limiting characteristics of the drug molecule, and protects it from enzymatic degradation. Once the NP has invaded the brain parenchyma, therapeutic drug release from the colloidal carrier may occur by desorption, diffusion through the NP matrix or polymer wall and or NP erosion [5,6]. Indeed, NPs have been shown to improve the brain distribution of drugs previously impermeable, i.e. dalargin, tubocurarine and doxorubicin [7– 9]. While improved brain delivery has been demonstrated, the mechanism of NP BBB circumvention is still theorized [6]. Potential methods of NP brain penetration include paracellular movement (after endothelial cell tight junction compromise), simple passive diffusion, transport and or endocytocis [5,6]. We have recently shown significant brain uptake of a novel NP formulation, comprised of emulsifying wax and Brij 78 [10], using the in-situ rat brain perfusion technique [10,11]. It was demonstrated that brain uptake of the NPs in-vivo and in-vitro occurred without any effect on significant baseline BBB parameters, i.e. cerebral perfusion flow, barrier integrity and permeability [12]. Considering: (1) the brain perfusion studies, (2) the lack of toxicity and (3) the relative short time ( < 60 s) for the NPs to penetrate the BBB, it appeared that the nanoparticles primarily penetrated the BBB by passive permeation. While brain distribution is critical for the success of NPs as a delivery system, the ability of the NPs to specifically target brain should also be considered. The proposed passive permeation of the NPs may also lead to increased peripheral organ distribution. Thus, to specifically target brain, a thiamine ligand was incorporated on the surface of the NPs. The targeting of NPs is not without precedent. Various types of NP targeting ligands have been employed including antibodies, peptides and vitamins [13,14]. Thiamine is a water-soluble micronutrient that is essential for normal cell function, growth and development. The consideration of thiamine as a cell specific ligand for targeted delivery can be rationalized since all eukaryotic cells have a specified transport mechanism for thiamine. In this regard, we previously studied the effectiveness of using the thiamine ligand in tumortargeting [15]. It was observed that thiamine-coated gadolinium NPs had specific association with human
breast cancer cells that expressed the thiamine transporters THTR1 and THTR2 [15]. Similarly, given the rich number of nutrient transporters at the BBB, the thiamine ligand should bind to the BBB thiamine transporter, subsequently increasing the number of NPs at the BBB interface. Further, based upon BBB thiamine transport capacity and kinetics [16,17], this nutrient transporter has been suggested as a brain drug delivery vector [18]. We hypothesize that the thiamine-coated NPs (thiamineNPs) will specifically favor brain uptake by either a facilitated transport mechanism or increased passive diffusion secondary to an increased concentration gradient of the NP located at the BBB interface due to association with the thiamine transporter. In the present study, thiamine-coated NPs were engineered directly from microemulsion precursors. The brain uptake and distribution of NPs with and with out thiamine as a brain targeting ligand were investigated.
2. Materials and methods 2.1. Materials Emulsifying wax (E. Wax) and DispoDialyzers MWCO 100 kDa were purchased from Spectrum Chemicals (New Brunswick, NJ). Polyoxyl 20stearyl ether (Brij 78) was obtained from Uniquema (Wilmington, DE). Distearoylphosphatidylethanolamine (DSPE)-PEG-NHS (MW 3350) was purchased from Shearwater Polymers (Hunsville, AL). Thiamine hydrochloride was purchased from Aldrich Chemicals (Milwaukee, WI). Sephadex G-75, Sepharose CL-4B, potassium ferricyanide, phosphate buffered saline (PBS), fetal bovine serum and sodium chloride were purchased from Sigma (St. Louis, MO). Mice (female; BALB/c) were purchased from Harlan (Indianapolis, IN). 2.2. Radiochemicals High specific activity [3H]thiamine (10 Ci/mmol, >98% purity) was obtained from American Radiolabeled Chemicals (St. Louis, MO). [14C]Sucrose (4.75 mCi/mmol) was obtained from Dupont-New England Nuclear (Boston, MA). 1-[3H]Hexadecanol (1 mCi/ml; radiochemical purity >96%) was pur-
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chased from Moravek Biochemicals (Brea, CA). For all experiments, deionized water was filtered through 0.22 Am filters (Nalgene International, Rochester, NY). Indium-111 chloride (2 mCi) in 0.05 M HCL was purchased from Perkin Elmer (Boston, MA). In each perfusion experiment, [3H]thiamine was dried prior to being dissolved in perfusion buffer, to remove volatile tritium contaminants including [3H]H2O. 2.3. Preparation of nanoparticles from microemulsion precursors Solid NPs were prepared from oil-in-water microemulsion precursors as described previously [19]. Briefly, 2 mg of emulsifying wax (oil phase) was accurately weighed, placed into a glass vial and melted on a hotplate. To the melted matrix at 55 jC, polyoxyl 20-stearyl ether (Brij 78; final concentration of 3 mM) was added under magnetic stirring. Water was added to make a final volume of 1 ml. The formation of oil-in-water microemulsion was verified by the clarity of the mixture and by photon correlation spectroscopy (PCS) at 55 jC using an N4 Plus Submicron Particle Sizer. Solid NPs were obtained by simple cooling the warm microemulsion to room temperature in one vessel. The cured NPs (E78 NPs; 2 mg/ml) were characterized based on size, size distribution and morphology. 2.4. Preparation of thiamine-coated nanoparticles A thiamine ligand was synthesized by chemically linking thiamine to DSPE via a PEG spacer (Mw 3350) as reported earlier [15]. Using a stock aqueous solution of thiamine ligand (thiamine-PEG-DSPE), 0.2% w/w thiamine ligand was added to cured NP suspensions (2 mg/ml) at 25 jC. The mixture was gently stirred for 4 h at 25 jC. The efficiency of thiamine attachment/adsorption was assessed by gel permeation chromatography (GPC) elution profiles using a Sepharose CL-4B column. Briefly, 80 Al of thiamine-coated NP suspensions were passed down the Sepharose CL-4B column (1.5 8 cm) using deionized water (0.22 Am filtered) as the mobile phase. The elution of thiamine-coated NPs and free thiamine ligand in all GPC fractions was detected by laser light scattering counts per second (CPS) and thiochrome assay. The GPC elution profiles of
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control NPs (without thiamine) and free thiamine ligand were obtained to serve as references. Based on the GPC elution profiles, the efficiency of thiamine ligand coating was calculated as the percentage of the ratio of the area under thiaminecoated NP profiles to the area under the total elution profiles. Calculation of the concentration of thiamine and total number of thiamine molecules used in coating NPs was based on coating efficiency data. 2.5. Characterization of thiamine-coated nanoparticles 2.5.1. Photon correlation spectroscopy (PCS) The particle size of thiamine-coated NPs was determined using an N4 Plus Sub-Micron Particle Sizer at 20 jC by scattering light at angle 90j for 180 s (Beckman Coulter, Miami, FL). Prior to particle size measurements, the NPs were diluted (1:10 v/v) with water, to ensure that the light scattering signal as indicated by the particle counts per second was within the sensitivity range of the instrument. 2.5.2. Gel permeation chromatography (GPC) To obtain the GPC elution profiles of NPs, 80 Al of NP suspension was passed down a Sephadex G-75 column (1.5 8 cm) using deionized water (0.22 Am filtered) as the mobile phase. The elution of thiaminecoated NPs was detected by laser light scattering counts per second and thiochrome assay using fluorescence spectroscopy (Hitachi Model F-2000). The thiochrome assay [15], involved oxidizing thiamine in each GPC fraction to thiochrome and subsequently measuring fluorescence intensity at 365 (excitation) and 445 nm (emission). 2.5.3. Transmission electron microscopy (TEM) The size and morphology of NPs were observed using a Philips Tecnai 12 electron microscope in the Imaging Facility Unit of the University of Kentucky. A carbon-coated 200-mesh copper specimen grid (Ted Pella, Redding, CA) was glow-discharged for 1.5 min. One drop of NP suspension was deposited on the grid and allowed to stand for 1.5 min after which any excess fluid was removed with filter paper. The grid was later stained with 1 drop of 1% uranyl acetate (0.22 Am filtered) for 30 s and any excess stain removed. The grids were allowed to dry for an
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additional 10 min before examination under the electron microscope. 2.6. Nanoparticle radiolabeling To allow for easy detection in the brain perfusion studies, the NP preparation method was modified as described earlier [10] to include a radiotracer in both the control NPs ([3H]NPs) and the thiamine coated NPs ([3H]thiamine-NPs). Briefly, a trace amount of [3H]hexadecanol (cetyl alcohol) was added to emulsifying wax prior to the formation of the microemulsion precursors at 55 jC. Nanoparticles containing entrapped [3H]hexadecanol were cured from warm microemulsions as described above. The entrapment efficiency of the radiotracer was f 100%. All final preparations had theoretical activities of 150 ACi per ml. 2.7. Perfusion procedure Initial experiments were focused on assessing uptake of [3H]thiamine-NPs and [3H]NPs into brain. The results obtained for [3H]NP were consistent with earlier observations by Koziara et al. [10]. Briefly, uptake was evaluated using the in-situ rat brain perfusion technique of Takasato et al. [11] with modifications described [20,21]. Perfusions of 5 – 120 s were used to determine initial brain uptake of the NPs. All studies were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Male Fischer-344 rats (220 – 330 g; Charles River Laboratories, Kingston, NY) were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneal). A PE-60 catheter filled with heparinized saline (100 units/ml) was placed into the left common carotid artery after ligation of the left external carotid, occipital and common carotid arteries. Common carotid artery ligation was accomplished caudal to the catheter implantation site. The pterygopalatine artery was left open during the experiments [21]. Rat rectal temperature was monitored and maintained at 37 jC by a heating pad connected to a feedback device (YSI Indicating Controller, Yellow Springs, OH). The catheter to the left common carotid artery was connected to a syringe containing buffered physiologic perfusion
fluid (containing [in mM]: NaCl 128, NaPO3 2.4, NaHCO3 29.0, KCl 4.2, CaCl 1.5, MgCl2 0.9, and Dglucose 9) with 1 ACi/ml [3H]NPs or [3H]thiamineNPs (final NP concentration f 20 Ag/ml) and 0.3 ACi/ml [14C]sucrose (to determine vascular volume). Perfusion fluid was filtered and warmed to 37 jC and gassed with 95% O2 and 5% CO2. The pH and osmolarity of this solution were c 7.35 and 290 mOsm, respectively, immediately prior to perfusion. The perfusion fluid was infused into the left carotid artery with an infusion pump for periods of 5– 120 s at 10 ml/min (Harvard Apparatus, South Natick, MA.). This perfusion rate was selected to maintain a carotid artery pressure of f 120 mm Hg [11]. Rats were decapitated and cerebral samples obtained as previously described [21]. Briefly, the brain was removed from the skull, and the perfused cerebral hemisphere dissected on ice after removal of the arachnoid membrane and meningeal vessels. Brain regions were placed in scintillation vials and weighed. In addition, two 50-Al aliquots of the perfusion fluid were transferred to a scintillation vial and weighed. The brain and perfusion fluid samples were then digested overnight at 50 jC in 1 ml of 1 M piperidine. Ten ml of Fisher Chemical scintillation cocktail (Beckman, Fullerton, CA) was added to each vial and the tracer contents assessed by dual-label liquid scintillation counting. Dual labeled scintillation counting of brain and perfusate samples were accomplished with correction for quench, background and efficiency. 2.8. Kinetic analysis Concentrations of NP tracer in brain and perfusion fluid are expressed as dpm/g brain or dpm/ml perfusion fluid, respectively. Blood – brain barrier [3H]NP brain uptake was determined by perfusion with [3H]thiamine-NPs and [3H]NPs in separate experiments for 5 – 120 s periods as described previously [10,11,20]. Specific time points for uptake evaluation between 45 and 120 s were: 45, 60, 90 and 120 s. Given the apparent linear uptake of the [3H]NPs in the time frames evaluated a unidirectional uptake transfer constant (Kin) was calculated from the following relationship to the linear portion of the uptake curve as described [20]: Q*=C* ¼ Kin T þ V0
ð1Þ
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where Q* is the quantity of [3H]tracer in brain (dpm/ g) at the end of perfusion, C* is the perfusion fluid concentration of [3H]NP (dpm/ml), T is the perfusion time (s) and V0 is the extrapolated intercept (T = 0 s; ‘‘vascular volume’’ in ml/g). Tracer trapped in the vascular space was accounted for by the subtraction of [14C]sucrose vascular volume. Given the [3H]thiamine-NPs had an apparent nonlinear initial brain uptake pattern, a calculated uptake transfer constant (Kin) and a brain efflux rate coefficient (kout) was estimated from the following relationship as described [20]: Q*=C* ¼ ðKin =kout Þð1 ekout T Þ
ð2Þ
where Q* is the quantity of [3H]tracer in brain (dpm/ g) at the end of perfusion, C* is the perfusion fluid concentration of [3H]thiamine (dpm/ml) and T is the perfusion time. Tracer trapped in the vascular space was accounted for by the subtraction of [14C]sucrose vascular volume. For determination of [3H]thiamine BBB uptake we calculated Kin by single point uptake experiments at 15 s in from the following relationship as described [20]: Kin ¼ ½Qtot Vv Cpf =ðCpf =T Þ
ð3Þ
where Qtot = Qbr + Qvas, the sum of the amount of thiamine remaining in the perfusate in the blood – brain vessels and the amount of thiamine that has penetrated into brain. Cerebral perfusion flow rate ( F) was determined in separate experiments as previously described [12]. Cpf is the perfusion fluid concentration of tracer thiamine and T is the net perfusion time. Kin values were then converted to apparent cerebrovascular permeability-surface area products (PA) using the Crone – Renkin equation [20]: PA ¼ Flnð1 Kin =FÞ
ð4Þ
2.9. In-vivo biodistribution studies in mice Biodistribution studies of thiamine-coated NPs were carried out in BALB/c mice (female; 16 g weight) after a 7-day acclimation period. The studies were approved by the University of Kentucky Animal Care and Use Committee in accordance with the NIH
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Guide for the Care and Use of laboratory animals. The two types of modifications that were carried out on thiamine-coated NP preparations used in the biodistribution experiments include: (1) addition of DSPEPEG (Mw 3000; in 3% w/w concentration) to emulsifying wax during the preparation of microemulsion precursors, and (2) entrapment of a trace quantity of indium-111 acetylacetonate ([111In]AcAc) in NPs. [111In]-labeled NPs allow fast and effective detection of NPs in various tissues using a gamma counter. After entrapment of the 111In-label, thiamine-coated NPs were prepared as described earlier (Section 2.5). The two types of NPs used in the biodistribution experiments were: (1) thiamine-coated NPs, and (2) control NPs (PEG-coated) that were not coated with the thiamine ligand. Prior to administration to mice, all NP preparations were diluted (1:1) with 0.9% sodium chloride solution (0.2 Am filtered). Each mouse was anesthetized and then injected (by tail vein administration) with a dose of 5 mg/kg of either thiamine-coated or PEG-coated NPs. At 2 or 6 h postinjection, the mice were sacrificed after a saline flush and visual inspection for apparent blood (prevented overestimation secondary to vascular contributions of radiolabel). The biodistribution of the [111In] radioactivity in tissues such as the blood, liver, lungs, kidneys, heart, spleen and brain (obtained from each mouse) was measured by a Cobra II auto gamma counter (Parkard BioScience, Meriden, CT). The residual radioactivity in the tail of each mouse was also measured to enable exact quantification of the injected dose. The total blood volume of a mouse was assumed to be 7.5% (v/w) of the total mouse [22,23]. 2.10. Statistical analysis Brain tissue data presented are from the frontal cerebral cortex unless otherwise specified. [3H]thiamine-NP and [3H]NP brain uptake over time were fit with non-linear regression and linear regression, respectively, using least squares analysis. One-way analysis of variance followed by a Bonferoni’s multiple comparison test were used for evaluation of the inhibition of brain uptake of [3H]thiamine. For all data, errors are reported as standard error of the mean, unless otherwise indicated (n = 3 – 5 for perfusion data, whereas biodistribution data n = 5 – 7). Differences were considered statistically significant a priori at
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the p < 0.05 level. (GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA).
3. Results 3.1. Preparation and characterization of thiaminecoated nanoparticles Microemulsions (oil-in-water) were used as precursors to obtain solid NPs [19]. To obtain thiaminecoated NPs, a thiamine ligand was synthesized using the procedure earlier described [15] and added to NP suspensions at 25 jC. The thiamine ligand contained a DSPE group and a PEG spacer. The attachment of thiamine ligand to NPs was confirmed by gel permeation chromatography. Based on the thiochrome assay, the NPs (2 mg/ml) contained 10 AM of thiamine. The TEM micrograph of thiamine-coated NPs is shown in Fig. 1, indicating that the NPs were approximately 100 nm in diameter. Additional measurements by laser light scattering showed that the mean diameter of thiamine-coated NPs was 67 F 8.2 nm with a polydispersity index of 0.2. The NPs were stable when incubated with PBS (pH 7.4) at 37 jC for 60 min (data not shown). 3.2. Nanoparticle brain uptake
Fig. 1. Transmission electron micrograph (TEM) showing the size and morphology of thiamine-coated nanoparticles.
Initial brain distribution parameters of [3H]NPs and [ H]thiamine-NP (1 ACi/20 Ag/ml) were evaluated using the rat brain perfusion method from 0 to 120 s [11]. In all experiments BBB integrity was verified with concurrent vascular volume measurements with [14C]sucrose. [14C]Sucrose vascular volumes ranged from 0.76 F 0.1 to 1.1 F 0.2 10 2 ml/g, consistent with previous in-situ NP brain perfusion data demonstrating an intact BBB [12]. Brain/perfusion fluid ratios (i.e. volume of distribution or ‘space’) were plotted as a function of time during the initial 45 s period and shown in Fig. 2. Given that the brain uptake of the [3H]NPs was apparently linear in this time frame, a unidirectional transfer coefficient (Kin) was calculated (3.37 F 0.2 10 3 ml/s/g) according to Eq. (1). In contrast to the [3H]NP linear uptake, the [3H]thiamine-NP demonstrated non-linear uptake during the initial 45 s uptake time. The [3H]thiamine-NP uptake data were fit to Eq.
(2), for a calculated Kin and kout of 7.11 F 1.9 10 3 ml/s/g and 5.6 F 2.4 10 2 s 1, respectively. To confirm the [3H]thiamine-NP efflux rate seen in the initial experiments washout studies were completed [16,21]. Briefly, the method consists of evaluating the tracer brain/perfusion ratios after: (1) a 45-s loading of either [3H]NP or [3H]thiamine-NP and (2) an immediate subsequent ‘‘wash’’ of NP/tracer free saline for periods of 15– 30 s. Fig. 3 shows brain perfusion ratios had no significant reduction during the 30 s tracer free wash period. Further, the efflux constant, based upon the linear regression of the slope, for either formulation ([ 3 H]NP: 5.0 F 0.82 10 4 s 1 ; [3H]thiamine-NP: 1.6 F 0.8 10 4 s 1) was not significant from a slope of zero. To verify that the NP thiamine ligand was associating with the thiamine BBB transporter, we calculated (Eqs. (3) and (4)) a PA for [3H]thiamine using
3
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Fig. 2. The time course of brain uptake for the [3H]NPs and [3H]thiamine-NPs during perfusion times of 5 to 45 s. The open circles represent the brain distribution volume of the [3H]thiamine-NPs in the presence of 50 AM thiamine. Data are frontal cerebral cortex; mean F S.E.M.; n = 3 – 5. Similar patterns were observed for other brain regions; data not shown. All values were corrected for vascular volume using concurrent [14C]sucrose distribution measurements.
Fig. 3. Time course of [3H]NPs and [3H]thiamine-NPs washout from brain (frontal cortex) after 45 s of brain perfusion uptake. Wash consisted of NP and tracer free saline. The linear regression of both slopes was not significant from zero ( p>0.05) indicating no efflux. All data represent mean F S.E.M. for frontal cortex; n = 3 – 5 for all points.
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Fig. 4. [3H]thiamine PA (cerebrovascular permeability-surface area product) in the presence of unlabeled thiamine (100 nM), the uncoated NP (20 Ag/ml; total NP dose f 200 Ag) and the thiamine-coated NPs (NP concentration was 20 ug/ml; thiamine ligand concentration was approximately 100 nM). An asterisk indicates that it differs significantly ( p < 0.05) from control. Data are for frontal cerebral cortex and represent the mean F S.E.M.; n = 3 – 5.
single time point uptake experiments for 15 s with results shown in Fig. 4. The calculated control [3H]thiamine PA (11.6 F 0.7 10 4 ml/s/g) and the approximate 20% inhibition (9.4 F 0.6 10 4 ml/s/ g) with 100 nM unlabeled thiamine agree with recently published in-situ rat brain perfusion data for [3H]thiamine [16]. Consistent with the above data, the presence of [3H]thiamine-NPs (total NP concentration of 20 Ag/ml with thiamine ligand concentration
of approximately 100 nM) resulted in inhibition (8.9 F 0.2 10 4 ml/s/g) similar to the presence of 100 nM thiamine. The uncoated NP had no apparent inhibition of [3H]thiamine BBB transport. To evaluate if the [3H]thiamine-NPs are accessing brain via the BBB thiamine transporter, we incorporated unlabeled thiamine into the perfusion fluid at concentrations (50 AM) that would completely inhibit BBB thiamine transport, and subsequently decrease
Fig. 5. Calculated transfer coefficients for both NPs during the 45 to 120 s perfusion time frame. An asterisk indicates that it differs significantly ( p < 0.05) from control. Data are for frontal cerebral cortex and represent the mean F S.E.M.; n = 3 – 5.
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[3H]thiamine-NP brain distribution [16]. Fig. 2 (empty circles and inset) shows brain distribution volume of [3H]thiamine-NP with 50 AM of thiamine incorporated into the perfusion fluid. The 15 s brain distribution volume was not significantly different from the value obtained in the absence of thiamine (0.13 F 0.08; 0.091 F 0.008 ml/g). However, in contrast to the expected decrease of brain distribution at 45 s, we observed a significant increase ( p < 0.05) of [3H]thiamine-NP brain distribution in the presence of 50 AM thiamine (0.12 F 0.02; 0.17 F 0.02 ml/g). Subsequent perfusion experiments to 120 s were completed to determine extended time frame brain uptake. In contrast to the non-linear and linear uptake patterns seen in early perfusion experiments, both the
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[3H]NP and the [3H]thiamine-NP had a linear brain uptake pattern from 45 to 120 s. The calculated Kin (Eq. (1)) during this time period for each NP is shown in Fig. 5. The [3H]thiamine-NPs had a significantly ( p < 0.05) increased Kin (9.8 F 1.1 10 3 ml/s/g) compared to the uncoated NPs (7.0 F 0.3 10 3 ml/s/g). 3.3. In-vivo biodistribution studies in mice The results of the biodistribution experiments in mice (BALB/c) are shown in Fig. 6A and B. Each mouse was injected with 5 mg/kg of NP suspensions in 0.9% sodium chloride. The results shown in Fig. 6A indicate that thiamine coating did not have a significant effect on final organ biodistribution ( p>0.05). The two
Fig. 6. (A) In-vivo biodistribution studies of (111In-labeled) thiamine-coated nanoparticles (THNP) and PEG-coated nanoparticles (CTRNP) in mice (BALB/c). (B) The amounts of radioactivity distributed to the brain of mice (BALB/c) at 2 and 6 h post-injection of (111In-labeled) thiamine-coated nanoparticles (THNP) and PEG-coated nanoparticles (CTRNP). Data represents the mean F S.D. (n = 5 – 7 mice).
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NPs had long circulating properties (Fig. 6A). Specifically, the thiamine-coated NPs had amounts of radioactivity in circulation at 2 and 6 h of 79% injected dose (ID) and 65% ID, respectively. The cumulative radioactivity distributed to reticuloendothelial system tissues (liver and spleen) were 13% ID and 22% ID at 2 and 6 h, respectively. NP stability was demonstrated by the low accumulated radioactivity in the lungs (Fig. 6A). In both NPs very low levels of radioactivity were observed in other tissues such as the lungs, kidneys and heart. The amounts of radioactivity distributed to the brain for the two types of NPs are shown in Fig. 6B. For both thiamine-coated and PEG-coated NPs, the average brain radioactivity levels for thiamine-coated NPs at 2 and 6 h were slightly less than 0.5% ID and did not differ significantly ( p>0.05) (Fig. 6B).
4. Discussion The results of the studies presented herein demonstrated: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of NPs with and without thiamine surface ligands, and (3) initial data suggesting mechanisms for NP brain entry. Comparison of the NP Kin demonstrated the [3H]thiamine-NPs associated with the BBB thiamine transporter and had increased brain uptake between 45 and 120 s. This data suggests that the thiamine ligand on the NPs may facilitate binding and/or association with BBB thiamine transporters. The relevance of this report is twofold; first, to our knowledge there is no other work that has specifically targeted NPs to brain in this manner, and second we have provided a highly sensitive kinetic analysis of NP movement at the BBB. The NPs used in this study were prepared using oil-in-water microemulsions as precursors. The ability to use microemulsions as precursors to engineer nanoparticles is in agreement with earlier studies [19]. The application of microemulsions as templates for NP production offers numerous advantages, demonstrated in this report, such as: (1) simplistic production of NPs ranging approximately 100 nm in diameter, (2) ease of microemulsion incorporation of hydrophobic compounds in the oil droplets (facilitating entrapment in cured NPs), and (3) inclusion of site-specific ligands in the NP preparations.
As shown in the TEM micrograph (Fig. 1), NP size averaged f 100 nm. The trend observed from laser light scattering also confirmed thiamine-coated NPs were f 100 nm with unimodal size distribution. Prior to brain perfusion studies, the stability of the NPs in relevant media was demonstrated (data not shown). Based on previous studies using liposomes and other macromolecules, we considered that the NP size of 100 nm as suitable for cell targeting and controlled release. The preparation technique also demonstrated significant versatility in that, for both the perfusion studies and biodistribution studies, different radiotracers were included in the NPs and entrapped at f 100% efficiency (data not shown) similar to previously published data [10]. The synthesized thiamine ligand has both DSPE groups and a PEG spacer. The DSPE hydrophobic groups were engineered to act as anchors and influence the insertion/adsorption of the thiamine ligand to the NP. The PEG spacer was included to facilitate flexibility and cell recognition of ligand on NP surface. Thiamine-coated NPs were obtained by adding 0.2% w/w thiamine ligand to NP suspensions at 25 jC. This NP formulation has been shown in-vitro to positively associate with human breast cancer cells expressing the thiamine transporters THTR1 and THTR2 [15]. To determine if the thiamine-coated NPs would similarly associate with BBB thiamine transporters we completed brain uptake evaluation for both the [3H]NPs and [3H]thiamine-NPs using the in-situ rat brain perfusion technique [11] (Fig. 2). The calculated transfer coefficient (Kin) based upon the initial 45 s linear uptake for the [3H]NPs is consistent with previously published in-situ NP perfusion data [10]. However, in contrast to the previous report, the [3H]thiamine-NPs had an initial brain distribution pattern that was non-linear in nature suggesting efflux or delayed brain penetration. Of interest, the placement of thiamine ligands on the NP created a brain uptake configuration consistent with [3H]thiamine brain uptake and efflux during insitu brain perfusions of less than 120 s [16]. Therefore, after evaluation of initial NP brain distribution we chose to evaluate [3H]thiamine-NP and [3H]NP efflux (Fig. 3) Specifically, the [3H]thiamine-NP complex contrasted [3H]thiamine BBB movement, in that there was no significant efflux during the washout
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study. However, similar to [3H]thiamine BBB movement there was no apparent BBB endothelial cell disassociation as seen by post-perfusion washouts resulting in an immediate reduction in the brain/ perfusion ratio [16,21]. Given the lack of efflux, we hypothesized the nonlinear [3H]thiamine-NP uptake pattern was secondary to: (1) facilitated but delayed transport into brain via the BBB thiamine transporter without subsequent efflux, and/or (2) the ligand –NP complex was associating with the carrier creating a subsequent delay in passive brain permeation. To determine if the NP was being transported across the BBB, experiments were completed to show substrate transporter association and saturability [20]. Therefore, we performed [3H]thiamine uptake experiments at 15 s with and without the presence of both NPs and unlabeled thiamine. The experimental time frame was based on previous [3H]thiamine BBB kinetic modeling (i.e. the time was long enough for [3H]thiamine brain detection yet was short enough to minimize apparent efflux) [16]. Fig. 4 shows addition of unlabeled thiamine (100 nM) to the perfusion fluid resulted in a reduction of PA consistent with previously published results [16]. Of major significance the uncoated NPs did not inhibit BBB [3H]thiamine transport yet the thiamine-NPs did (thiamine concentration f 100 nM). The ability of the latter NP type to inhibit BBB uptake [3H]thiamine strongly suggests NP complex association with thiamine BBB transporter proteins. To determine if the [3H]thiamine-NP was accessing brain via the BBB thiamine transporter, we evaluated the saturability of the process by the incorporation of unlabeled thiamine (50 AM) into the perfusion fluid. If transport occurs this concentration will completely inhibit BBB [3H]thiamine-NP brain distribution via the respective carrier [16]. In contrast to the expected finding (i.e. inhibiting brain penetration of [3H]thiamine-NP with unlabeled thiamine), Fig. 2 (inset) shows the presence of unlabeled thiamine resulted in a significant increase of [3H]thiamine-NP brain distribution. This data implies that addition of the thiamine ligand to the NP does not result in BBB penetration via facilitated thiamine BBB transport but rather the ligand – NP complex is associating with the thiamine BBB transporter with a subsequent delay in passive brain permeation.
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After determining the [3H]thiamine-NP was associating with the BBB thiamine transporter, we hypothesized there may be accumulation of the thiaminecoated NPs at the BBB. Potentially, NP accumulation at the BBB may lead to improved brain uptake by increasing the concentration gradient. To assess this hypothesis, we extended perfusion time frames to 60, 75, 90 and 120 s. In contrast to the non-linear and linear brain uptake pattern seen in the initial 45 s time frame, both NPs had an apparent linear brain uptake profile during the extended perfusions. Fig. 5 shows the uptake transfer coefficient (Kin) for [3H]thiamineNPs is significantly greater than the [3H]NPs during the perfusion 45 to 120 s time frame. This data further supports the hypothesis of the thiamine ligand associating with the thiamine BBB transporter may improve overall NP brain uptake. To assess the improved NP brain uptake we completed biodistribution studies. The NPs were modified to contain a DSPE-PEG spacer based upon: (1) retention of the cured NPs in blood [24] and (2) PEG-coated macromolecules display specific affinity for brain endothelial cells [25]. As shown in Fig. 6A, both thiamine and PEG-coated (control) NPs had long circulating properties most likely due to PEG molecules preventing or at least minimizing reticuloendothelial NP recognition. It is also noteworthy there was an apparent lack of NP aggregation (leading to rapid lung deposition) as demonstrated by the low levels of radioactivity observed in the lungs (Fig. 6A). For both thiamine-coated and PEG-coated NPs, low levels of radioactivity were distributed to the brain at the time points studied. In the biodistribution studies (Fig. 6B), thiamine ligand coating on NPs did not have a significant effect on brain radioactivity levels (in comparison to control) possibly due to a number of factors including: (1) addition of 0.2% w/w of thiamine ligand may result in low and insufficient density of thiamine ligand coating on NPs in-vivo, (2) brain distribution at extended time points (beyond 6 h) were not studied, (3) normal blood thiamine levels in mice may competitively inhibit brain uptake of thiamine-coated NPs, (4) the thiamine NP complex may interact with erythrocyte thiamine transporters and (5) in-vivo shear flow and dilution effects could diminish the amount of thiamine-coated NP association with BBB.
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In summary, the results presented herein indicate the addition of a thiamine ligand to the NPs causes association with the BBB thiamine transporter. This association may create an accumulation of NPs at the BBB, which ultimately increases brain uptake during perfusion time frames. Of major importance, the blood retention properties of both NPs may increase their potential utility in site-specific and controlled drug delivery.
[10]
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Acknowledgements The research was supported, in part, by NIH/NIBIB grant EB00531-01 to Dr. Allen and Dr. Mumper. The authors gratefully acknowledge the Cancer Biology Research Center at the Texas Tech University Health Sciences Center School of Pharmacy, and an American Foundation for Pharmaceutical Education pre-doctoral fellowship for financial support. References [1] W.M. Pardridge, Drug and gene delivery to the brain: the vascular route, Neuron 36 (4) (2002) 555 – 558. [2] M.W. Brightman, Morphology of blood – brain interfaces, Exp. Eye Res. 25 (1977) 1 – 25 (Suppl.). [3] W.M. Pardridge, Brain drug targeting and gene technologies, Jpn. J. Pharmacol. 87 (2) (2001) 97 – 103. [4] T. Terasaki, Development of brain efflux index (BEI) method and its application to the blood – brain barrier efflux transport study, in: W.M. Pardridge (Ed.), Introduction to the Blood – Brain Barrier, Cambridge Univ. Press, New York, 1998, pp. 24 – 31. [5] P.R. Lockman, R.J. Mumper, M.A. Khan, D.D. Allen, Nanoparticle technology for drug delivery across the blood – brain barrier, Drug Dev. Ind. Pharm. 28 (1) (2002) 1 – 12. [6] J. Kreuter, R.N. Alyautdin, Using nanoparticles to target drugs to the central nervous system, in: D.J. Begley, M.W. Bradbury, J. Kreuter (Eds.), The Blood – Brain Barrier and Drug Delivery to the CNS, Marcel Dekker, New York, 2000, pp. 205 – 223. [7] A. Gulyaev, S.E. Gelperina, I.N. Skidan, A.S. Antropov, G.Y. Kivman, J. Kreuter, Significant transport of doxorubicin into the brain with polysorbate 80 coated nanoparticles, Pharm. Res. 16 (10) (1999) 1564 – 1569. [8] U. Schroder, B.A. Sabel, Nanoparticles, a drug carrier system to pass the blood – brain barrier, permit central analgesic effects of i.v. dalargin injections, Brain Res. 710 (1 – 2) (1996) 121 – 124. [9] R.N. Alyautidin, E.B. Tezikov, P.D. Ramage, A. Kharkevich, D.J. Begly, J. Kreuter, Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 80-coated poly
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Brain uptake of thiamine-coated nanoparticles Paul R. Lockman a, Moses O. Oyewumi b, Joanna M. Koziara b, Karen E. Roder a, Russell J. Mumper b, David D. Allen a,* a
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, 1300 So. Coulter Drive, Amarillo, TX 79106-1712, USA b Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA Received 15 May 2003; accepted 18 August 2003
Abstract Recently, a novel nanoparticle (NP) comprised of emulsifying wax and Brij 78 was shown to have significant brain uptake using the in-situ rat brain perfusion technique. To further these studies and to specifically target brain, we have incorporated thiamine as a surface ligand on the nanoparticles. Solid nanoparticles were prepared from oil-in-water microemulsion precursors. Nanoparticles were radiolabeled and a thiamine ligand (thiamine linked to distearoylphosphatidylethanolamine via a polyethylene glycol spacer) was coated on the surface of the nanoparticles. Initial experiments focused on assessing uptake of [3H]nanoparticles with and without thiamine surface ligands. Biodistribution nanoparticle studies were also carried out in BALB/c mice. The results showed: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of nanoparticles with and without the thiamine surface ligands, and (3) initial data suggesting mechanisms for nanoparticle brain entry. Comparison of NP brain uptake demonstrated that the thiamine-coated nanoparticle associated with the blood – brain barrier (BBB) thiamine transporter and had an increased Kin between 45 and 120 s (thiamine coated NP 9.8 F 1.1 10 3 ml/s/g versus uncoated NPs; 7.0 F 0.3 10 3 ml/s/g). It was concluded that the thiamine ligand facilitated binding and/or association with blood – brain barrier thiamine transporters, which may be a viable mechanism for nanoparticle mediated brain drug delivery. D 2003 Elsevier B.V. All rights reserved. Keywords: Microemulsions; Blood – brain barrier; Emulsifying wax; Targeting; Biodistribution
1. Introduction Effective brain drug delivery is limited by the blood – brain barrier (BBB). This interface between plasma and brain consists of both brain microvascular endothelium and the surrounding glia. The BBB * Corresponding author. Tel.: +1-806-356-4000x286; fax: +1806-356-4034. E-mail address: [email protected] (D.D. Allen). 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.08.006
significantly restricts water-soluble, charged and high molecular weight therapeutics to the vascular space while allowing brain parenchyma penetration of small and/or lipophilic molecules [1]. Mechanisms of permeability regulation include: (1) microvascular endothelial tight junctions [2], (2) enzymatic regulation [3], and (3) active brain efflux [4]. While multiple strategies have been employed to circumvent the BBB, an emerging approach is the use of nanoparticles (NPs). The composition of the nanoparticles can be adjusted
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to allow almost any therapeutic drug to be adsorbed or entrapped. This method effectively disguises the membrane barrier limiting characteristics of the drug molecule, and protects it from enzymatic degradation. Once the NP has invaded the brain parenchyma, therapeutic drug release from the colloidal carrier may occur by desorption, diffusion through the NP matrix or polymer wall and or NP erosion [5,6]. Indeed, NPs have been shown to improve the brain distribution of drugs previously impermeable, i.e. dalargin, tubocurarine and doxorubicin [7– 9]. While improved brain delivery has been demonstrated, the mechanism of NP BBB circumvention is still theorized [6]. Potential methods of NP brain penetration include paracellular movement (after endothelial cell tight junction compromise), simple passive diffusion, transport and or endocytocis [5,6]. We have recently shown significant brain uptake of a novel NP formulation, comprised of emulsifying wax and Brij 78 [10], using the in-situ rat brain perfusion technique [10,11]. It was demonstrated that brain uptake of the NPs in-vivo and in-vitro occurred without any effect on significant baseline BBB parameters, i.e. cerebral perfusion flow, barrier integrity and permeability [12]. Considering: (1) the brain perfusion studies, (2) the lack of toxicity and (3) the relative short time ( < 60 s) for the NPs to penetrate the BBB, it appeared that the nanoparticles primarily penetrated the BBB by passive permeation. While brain distribution is critical for the success of NPs as a delivery system, the ability of the NPs to specifically target brain should also be considered. The proposed passive permeation of the NPs may also lead to increased peripheral organ distribution. Thus, to specifically target brain, a thiamine ligand was incorporated on the surface of the NPs. The targeting of NPs is not without precedent. Various types of NP targeting ligands have been employed including antibodies, peptides and vitamins [13,14]. Thiamine is a water-soluble micronutrient that is essential for normal cell function, growth and development. The consideration of thiamine as a cell specific ligand for targeted delivery can be rationalized since all eukaryotic cells have a specified transport mechanism for thiamine. In this regard, we previously studied the effectiveness of using the thiamine ligand in tumortargeting [15]. It was observed that thiamine-coated gadolinium NPs had specific association with human
breast cancer cells that expressed the thiamine transporters THTR1 and THTR2 [15]. Similarly, given the rich number of nutrient transporters at the BBB, the thiamine ligand should bind to the BBB thiamine transporter, subsequently increasing the number of NPs at the BBB interface. Further, based upon BBB thiamine transport capacity and kinetics [16,17], this nutrient transporter has been suggested as a brain drug delivery vector [18]. We hypothesize that the thiamine-coated NPs (thiamineNPs) will specifically favor brain uptake by either a facilitated transport mechanism or increased passive diffusion secondary to an increased concentration gradient of the NP located at the BBB interface due to association with the thiamine transporter. In the present study, thiamine-coated NPs were engineered directly from microemulsion precursors. The brain uptake and distribution of NPs with and with out thiamine as a brain targeting ligand were investigated.
2. Materials and methods 2.1. Materials Emulsifying wax (E. Wax) and DispoDialyzers MWCO 100 kDa were purchased from Spectrum Chemicals (New Brunswick, NJ). Polyoxyl 20stearyl ether (Brij 78) was obtained from Uniquema (Wilmington, DE). Distearoylphosphatidylethanolamine (DSPE)-PEG-NHS (MW 3350) was purchased from Shearwater Polymers (Hunsville, AL). Thiamine hydrochloride was purchased from Aldrich Chemicals (Milwaukee, WI). Sephadex G-75, Sepharose CL-4B, potassium ferricyanide, phosphate buffered saline (PBS), fetal bovine serum and sodium chloride were purchased from Sigma (St. Louis, MO). Mice (female; BALB/c) were purchased from Harlan (Indianapolis, IN). 2.2. Radiochemicals High specific activity [3H]thiamine (10 Ci/mmol, >98% purity) was obtained from American Radiolabeled Chemicals (St. Louis, MO). [14C]Sucrose (4.75 mCi/mmol) was obtained from Dupont-New England Nuclear (Boston, MA). 1-[3H]Hexadecanol (1 mCi/ml; radiochemical purity >96%) was pur-
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chased from Moravek Biochemicals (Brea, CA). For all experiments, deionized water was filtered through 0.22 Am filters (Nalgene International, Rochester, NY). Indium-111 chloride (2 mCi) in 0.05 M HCL was purchased from Perkin Elmer (Boston, MA). In each perfusion experiment, [3H]thiamine was dried prior to being dissolved in perfusion buffer, to remove volatile tritium contaminants including [3H]H2O. 2.3. Preparation of nanoparticles from microemulsion precursors Solid NPs were prepared from oil-in-water microemulsion precursors as described previously [19]. Briefly, 2 mg of emulsifying wax (oil phase) was accurately weighed, placed into a glass vial and melted on a hotplate. To the melted matrix at 55 jC, polyoxyl 20-stearyl ether (Brij 78; final concentration of 3 mM) was added under magnetic stirring. Water was added to make a final volume of 1 ml. The formation of oil-in-water microemulsion was verified by the clarity of the mixture and by photon correlation spectroscopy (PCS) at 55 jC using an N4 Plus Submicron Particle Sizer. Solid NPs were obtained by simple cooling the warm microemulsion to room temperature in one vessel. The cured NPs (E78 NPs; 2 mg/ml) were characterized based on size, size distribution and morphology. 2.4. Preparation of thiamine-coated nanoparticles A thiamine ligand was synthesized by chemically linking thiamine to DSPE via a PEG spacer (Mw 3350) as reported earlier [15]. Using a stock aqueous solution of thiamine ligand (thiamine-PEG-DSPE), 0.2% w/w thiamine ligand was added to cured NP suspensions (2 mg/ml) at 25 jC. The mixture was gently stirred for 4 h at 25 jC. The efficiency of thiamine attachment/adsorption was assessed by gel permeation chromatography (GPC) elution profiles using a Sepharose CL-4B column. Briefly, 80 Al of thiamine-coated NP suspensions were passed down the Sepharose CL-4B column (1.5 8 cm) using deionized water (0.22 Am filtered) as the mobile phase. The elution of thiamine-coated NPs and free thiamine ligand in all GPC fractions was detected by laser light scattering counts per second (CPS) and thiochrome assay. The GPC elution profiles of
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control NPs (without thiamine) and free thiamine ligand were obtained to serve as references. Based on the GPC elution profiles, the efficiency of thiamine ligand coating was calculated as the percentage of the ratio of the area under thiaminecoated NP profiles to the area under the total elution profiles. Calculation of the concentration of thiamine and total number of thiamine molecules used in coating NPs was based on coating efficiency data. 2.5. Characterization of thiamine-coated nanoparticles 2.5.1. Photon correlation spectroscopy (PCS) The particle size of thiamine-coated NPs was determined using an N4 Plus Sub-Micron Particle Sizer at 20 jC by scattering light at angle 90j for 180 s (Beckman Coulter, Miami, FL). Prior to particle size measurements, the NPs were diluted (1:10 v/v) with water, to ensure that the light scattering signal as indicated by the particle counts per second was within the sensitivity range of the instrument. 2.5.2. Gel permeation chromatography (GPC) To obtain the GPC elution profiles of NPs, 80 Al of NP suspension was passed down a Sephadex G-75 column (1.5 8 cm) using deionized water (0.22 Am filtered) as the mobile phase. The elution of thiaminecoated NPs was detected by laser light scattering counts per second and thiochrome assay using fluorescence spectroscopy (Hitachi Model F-2000). The thiochrome assay [15], involved oxidizing thiamine in each GPC fraction to thiochrome and subsequently measuring fluorescence intensity at 365 (excitation) and 445 nm (emission). 2.5.3. Transmission electron microscopy (TEM) The size and morphology of NPs were observed using a Philips Tecnai 12 electron microscope in the Imaging Facility Unit of the University of Kentucky. A carbon-coated 200-mesh copper specimen grid (Ted Pella, Redding, CA) was glow-discharged for 1.5 min. One drop of NP suspension was deposited on the grid and allowed to stand for 1.5 min after which any excess fluid was removed with filter paper. The grid was later stained with 1 drop of 1% uranyl acetate (0.22 Am filtered) for 30 s and any excess stain removed. The grids were allowed to dry for an
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additional 10 min before examination under the electron microscope. 2.6. Nanoparticle radiolabeling To allow for easy detection in the brain perfusion studies, the NP preparation method was modified as described earlier [10] to include a radiotracer in both the control NPs ([3H]NPs) and the thiamine coated NPs ([3H]thiamine-NPs). Briefly, a trace amount of [3H]hexadecanol (cetyl alcohol) was added to emulsifying wax prior to the formation of the microemulsion precursors at 55 jC. Nanoparticles containing entrapped [3H]hexadecanol were cured from warm microemulsions as described above. The entrapment efficiency of the radiotracer was f 100%. All final preparations had theoretical activities of 150 ACi per ml. 2.7. Perfusion procedure Initial experiments were focused on assessing uptake of [3H]thiamine-NPs and [3H]NPs into brain. The results obtained for [3H]NP were consistent with earlier observations by Koziara et al. [10]. Briefly, uptake was evaluated using the in-situ rat brain perfusion technique of Takasato et al. [11] with modifications described [20,21]. Perfusions of 5 – 120 s were used to determine initial brain uptake of the NPs. All studies were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Male Fischer-344 rats (220 – 330 g; Charles River Laboratories, Kingston, NY) were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneal). A PE-60 catheter filled with heparinized saline (100 units/ml) was placed into the left common carotid artery after ligation of the left external carotid, occipital and common carotid arteries. Common carotid artery ligation was accomplished caudal to the catheter implantation site. The pterygopalatine artery was left open during the experiments [21]. Rat rectal temperature was monitored and maintained at 37 jC by a heating pad connected to a feedback device (YSI Indicating Controller, Yellow Springs, OH). The catheter to the left common carotid artery was connected to a syringe containing buffered physiologic perfusion
fluid (containing [in mM]: NaCl 128, NaPO3 2.4, NaHCO3 29.0, KCl 4.2, CaCl 1.5, MgCl2 0.9, and Dglucose 9) with 1 ACi/ml [3H]NPs or [3H]thiamineNPs (final NP concentration f 20 Ag/ml) and 0.3 ACi/ml [14C]sucrose (to determine vascular volume). Perfusion fluid was filtered and warmed to 37 jC and gassed with 95% O2 and 5% CO2. The pH and osmolarity of this solution were c 7.35 and 290 mOsm, respectively, immediately prior to perfusion. The perfusion fluid was infused into the left carotid artery with an infusion pump for periods of 5– 120 s at 10 ml/min (Harvard Apparatus, South Natick, MA.). This perfusion rate was selected to maintain a carotid artery pressure of f 120 mm Hg [11]. Rats were decapitated and cerebral samples obtained as previously described [21]. Briefly, the brain was removed from the skull, and the perfused cerebral hemisphere dissected on ice after removal of the arachnoid membrane and meningeal vessels. Brain regions were placed in scintillation vials and weighed. In addition, two 50-Al aliquots of the perfusion fluid were transferred to a scintillation vial and weighed. The brain and perfusion fluid samples were then digested overnight at 50 jC in 1 ml of 1 M piperidine. Ten ml of Fisher Chemical scintillation cocktail (Beckman, Fullerton, CA) was added to each vial and the tracer contents assessed by dual-label liquid scintillation counting. Dual labeled scintillation counting of brain and perfusate samples were accomplished with correction for quench, background and efficiency. 2.8. Kinetic analysis Concentrations of NP tracer in brain and perfusion fluid are expressed as dpm/g brain or dpm/ml perfusion fluid, respectively. Blood – brain barrier [3H]NP brain uptake was determined by perfusion with [3H]thiamine-NPs and [3H]NPs in separate experiments for 5 – 120 s periods as described previously [10,11,20]. Specific time points for uptake evaluation between 45 and 120 s were: 45, 60, 90 and 120 s. Given the apparent linear uptake of the [3H]NPs in the time frames evaluated a unidirectional uptake transfer constant (Kin) was calculated from the following relationship to the linear portion of the uptake curve as described [20]: Q*=C* ¼ Kin T þ V0
ð1Þ
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where Q* is the quantity of [3H]tracer in brain (dpm/ g) at the end of perfusion, C* is the perfusion fluid concentration of [3H]NP (dpm/ml), T is the perfusion time (s) and V0 is the extrapolated intercept (T = 0 s; ‘‘vascular volume’’ in ml/g). Tracer trapped in the vascular space was accounted for by the subtraction of [14C]sucrose vascular volume. Given the [3H]thiamine-NPs had an apparent nonlinear initial brain uptake pattern, a calculated uptake transfer constant (Kin) and a brain efflux rate coefficient (kout) was estimated from the following relationship as described [20]: Q*=C* ¼ ðKin =kout Þð1 ekout T Þ
ð2Þ
where Q* is the quantity of [3H]tracer in brain (dpm/ g) at the end of perfusion, C* is the perfusion fluid concentration of [3H]thiamine (dpm/ml) and T is the perfusion time. Tracer trapped in the vascular space was accounted for by the subtraction of [14C]sucrose vascular volume. For determination of [3H]thiamine BBB uptake we calculated Kin by single point uptake experiments at 15 s in from the following relationship as described [20]: Kin ¼ ½Qtot Vv Cpf =ðCpf =T Þ
ð3Þ
where Qtot = Qbr + Qvas, the sum of the amount of thiamine remaining in the perfusate in the blood – brain vessels and the amount of thiamine that has penetrated into brain. Cerebral perfusion flow rate ( F) was determined in separate experiments as previously described [12]. Cpf is the perfusion fluid concentration of tracer thiamine and T is the net perfusion time. Kin values were then converted to apparent cerebrovascular permeability-surface area products (PA) using the Crone – Renkin equation [20]: PA ¼ Flnð1 Kin =FÞ
ð4Þ
2.9. In-vivo biodistribution studies in mice Biodistribution studies of thiamine-coated NPs were carried out in BALB/c mice (female; 16 g weight) after a 7-day acclimation period. The studies were approved by the University of Kentucky Animal Care and Use Committee in accordance with the NIH
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Guide for the Care and Use of laboratory animals. The two types of modifications that were carried out on thiamine-coated NP preparations used in the biodistribution experiments include: (1) addition of DSPEPEG (Mw 3000; in 3% w/w concentration) to emulsifying wax during the preparation of microemulsion precursors, and (2) entrapment of a trace quantity of indium-111 acetylacetonate ([111In]AcAc) in NPs. [111In]-labeled NPs allow fast and effective detection of NPs in various tissues using a gamma counter. After entrapment of the 111In-label, thiamine-coated NPs were prepared as described earlier (Section 2.5). The two types of NPs used in the biodistribution experiments were: (1) thiamine-coated NPs, and (2) control NPs (PEG-coated) that were not coated with the thiamine ligand. Prior to administration to mice, all NP preparations were diluted (1:1) with 0.9% sodium chloride solution (0.2 Am filtered). Each mouse was anesthetized and then injected (by tail vein administration) with a dose of 5 mg/kg of either thiamine-coated or PEG-coated NPs. At 2 or 6 h postinjection, the mice were sacrificed after a saline flush and visual inspection for apparent blood (prevented overestimation secondary to vascular contributions of radiolabel). The biodistribution of the [111In] radioactivity in tissues such as the blood, liver, lungs, kidneys, heart, spleen and brain (obtained from each mouse) was measured by a Cobra II auto gamma counter (Parkard BioScience, Meriden, CT). The residual radioactivity in the tail of each mouse was also measured to enable exact quantification of the injected dose. The total blood volume of a mouse was assumed to be 7.5% (v/w) of the total mouse [22,23]. 2.10. Statistical analysis Brain tissue data presented are from the frontal cerebral cortex unless otherwise specified. [3H]thiamine-NP and [3H]NP brain uptake over time were fit with non-linear regression and linear regression, respectively, using least squares analysis. One-way analysis of variance followed by a Bonferoni’s multiple comparison test were used for evaluation of the inhibition of brain uptake of [3H]thiamine. For all data, errors are reported as standard error of the mean, unless otherwise indicated (n = 3 – 5 for perfusion data, whereas biodistribution data n = 5 – 7). Differences were considered statistically significant a priori at
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the p < 0.05 level. (GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego, CA).
3. Results 3.1. Preparation and characterization of thiaminecoated nanoparticles Microemulsions (oil-in-water) were used as precursors to obtain solid NPs [19]. To obtain thiaminecoated NPs, a thiamine ligand was synthesized using the procedure earlier described [15] and added to NP suspensions at 25 jC. The thiamine ligand contained a DSPE group and a PEG spacer. The attachment of thiamine ligand to NPs was confirmed by gel permeation chromatography. Based on the thiochrome assay, the NPs (2 mg/ml) contained 10 AM of thiamine. The TEM micrograph of thiamine-coated NPs is shown in Fig. 1, indicating that the NPs were approximately 100 nm in diameter. Additional measurements by laser light scattering showed that the mean diameter of thiamine-coated NPs was 67 F 8.2 nm with a polydispersity index of 0.2. The NPs were stable when incubated with PBS (pH 7.4) at 37 jC for 60 min (data not shown). 3.2. Nanoparticle brain uptake
Fig. 1. Transmission electron micrograph (TEM) showing the size and morphology of thiamine-coated nanoparticles.
Initial brain distribution parameters of [3H]NPs and [ H]thiamine-NP (1 ACi/20 Ag/ml) were evaluated using the rat brain perfusion method from 0 to 120 s [11]. In all experiments BBB integrity was verified with concurrent vascular volume measurements with [14C]sucrose. [14C]Sucrose vascular volumes ranged from 0.76 F 0.1 to 1.1 F 0.2 10 2 ml/g, consistent with previous in-situ NP brain perfusion data demonstrating an intact BBB [12]. Brain/perfusion fluid ratios (i.e. volume of distribution or ‘space’) were plotted as a function of time during the initial 45 s period and shown in Fig. 2. Given that the brain uptake of the [3H]NPs was apparently linear in this time frame, a unidirectional transfer coefficient (Kin) was calculated (3.37 F 0.2 10 3 ml/s/g) according to Eq. (1). In contrast to the [3H]NP linear uptake, the [3H]thiamine-NP demonstrated non-linear uptake during the initial 45 s uptake time. The [3H]thiamine-NP uptake data were fit to Eq.
(2), for a calculated Kin and kout of 7.11 F 1.9 10 3 ml/s/g and 5.6 F 2.4 10 2 s 1, respectively. To confirm the [3H]thiamine-NP efflux rate seen in the initial experiments washout studies were completed [16,21]. Briefly, the method consists of evaluating the tracer brain/perfusion ratios after: (1) a 45-s loading of either [3H]NP or [3H]thiamine-NP and (2) an immediate subsequent ‘‘wash’’ of NP/tracer free saline for periods of 15– 30 s. Fig. 3 shows brain perfusion ratios had no significant reduction during the 30 s tracer free wash period. Further, the efflux constant, based upon the linear regression of the slope, for either formulation ([ 3 H]NP: 5.0 F 0.82 10 4 s 1 ; [3H]thiamine-NP: 1.6 F 0.8 10 4 s 1) was not significant from a slope of zero. To verify that the NP thiamine ligand was associating with the thiamine BBB transporter, we calculated (Eqs. (3) and (4)) a PA for [3H]thiamine using
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Fig. 2. The time course of brain uptake for the [3H]NPs and [3H]thiamine-NPs during perfusion times of 5 to 45 s. The open circles represent the brain distribution volume of the [3H]thiamine-NPs in the presence of 50 AM thiamine. Data are frontal cerebral cortex; mean F S.E.M.; n = 3 – 5. Similar patterns were observed for other brain regions; data not shown. All values were corrected for vascular volume using concurrent [14C]sucrose distribution measurements.
Fig. 3. Time course of [3H]NPs and [3H]thiamine-NPs washout from brain (frontal cortex) after 45 s of brain perfusion uptake. Wash consisted of NP and tracer free saline. The linear regression of both slopes was not significant from zero ( p>0.05) indicating no efflux. All data represent mean F S.E.M. for frontal cortex; n = 3 – 5 for all points.
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Fig. 4. [3H]thiamine PA (cerebrovascular permeability-surface area product) in the presence of unlabeled thiamine (100 nM), the uncoated NP (20 Ag/ml; total NP dose f 200 Ag) and the thiamine-coated NPs (NP concentration was 20 ug/ml; thiamine ligand concentration was approximately 100 nM). An asterisk indicates that it differs significantly ( p < 0.05) from control. Data are for frontal cerebral cortex and represent the mean F S.E.M.; n = 3 – 5.
single time point uptake experiments for 15 s with results shown in Fig. 4. The calculated control [3H]thiamine PA (11.6 F 0.7 10 4 ml/s/g) and the approximate 20% inhibition (9.4 F 0.6 10 4 ml/s/ g) with 100 nM unlabeled thiamine agree with recently published in-situ rat brain perfusion data for [3H]thiamine [16]. Consistent with the above data, the presence of [3H]thiamine-NPs (total NP concentration of 20 Ag/ml with thiamine ligand concentration
of approximately 100 nM) resulted in inhibition (8.9 F 0.2 10 4 ml/s/g) similar to the presence of 100 nM thiamine. The uncoated NP had no apparent inhibition of [3H]thiamine BBB transport. To evaluate if the [3H]thiamine-NPs are accessing brain via the BBB thiamine transporter, we incorporated unlabeled thiamine into the perfusion fluid at concentrations (50 AM) that would completely inhibit BBB thiamine transport, and subsequently decrease
Fig. 5. Calculated transfer coefficients for both NPs during the 45 to 120 s perfusion time frame. An asterisk indicates that it differs significantly ( p < 0.05) from control. Data are for frontal cerebral cortex and represent the mean F S.E.M.; n = 3 – 5.
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[3H]thiamine-NP brain distribution [16]. Fig. 2 (empty circles and inset) shows brain distribution volume of [3H]thiamine-NP with 50 AM of thiamine incorporated into the perfusion fluid. The 15 s brain distribution volume was not significantly different from the value obtained in the absence of thiamine (0.13 F 0.08; 0.091 F 0.008 ml/g). However, in contrast to the expected decrease of brain distribution at 45 s, we observed a significant increase ( p < 0.05) of [3H]thiamine-NP brain distribution in the presence of 50 AM thiamine (0.12 F 0.02; 0.17 F 0.02 ml/g). Subsequent perfusion experiments to 120 s were completed to determine extended time frame brain uptake. In contrast to the non-linear and linear uptake patterns seen in early perfusion experiments, both the
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[3H]NP and the [3H]thiamine-NP had a linear brain uptake pattern from 45 to 120 s. The calculated Kin (Eq. (1)) during this time period for each NP is shown in Fig. 5. The [3H]thiamine-NPs had a significantly ( p < 0.05) increased Kin (9.8 F 1.1 10 3 ml/s/g) compared to the uncoated NPs (7.0 F 0.3 10 3 ml/s/g). 3.3. In-vivo biodistribution studies in mice The results of the biodistribution experiments in mice (BALB/c) are shown in Fig. 6A and B. Each mouse was injected with 5 mg/kg of NP suspensions in 0.9% sodium chloride. The results shown in Fig. 6A indicate that thiamine coating did not have a significant effect on final organ biodistribution ( p>0.05). The two
Fig. 6. (A) In-vivo biodistribution studies of (111In-labeled) thiamine-coated nanoparticles (THNP) and PEG-coated nanoparticles (CTRNP) in mice (BALB/c). (B) The amounts of radioactivity distributed to the brain of mice (BALB/c) at 2 and 6 h post-injection of (111In-labeled) thiamine-coated nanoparticles (THNP) and PEG-coated nanoparticles (CTRNP). Data represents the mean F S.D. (n = 5 – 7 mice).
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NPs had long circulating properties (Fig. 6A). Specifically, the thiamine-coated NPs had amounts of radioactivity in circulation at 2 and 6 h of 79% injected dose (ID) and 65% ID, respectively. The cumulative radioactivity distributed to reticuloendothelial system tissues (liver and spleen) were 13% ID and 22% ID at 2 and 6 h, respectively. NP stability was demonstrated by the low accumulated radioactivity in the lungs (Fig. 6A). In both NPs very low levels of radioactivity were observed in other tissues such as the lungs, kidneys and heart. The amounts of radioactivity distributed to the brain for the two types of NPs are shown in Fig. 6B. For both thiamine-coated and PEG-coated NPs, the average brain radioactivity levels for thiamine-coated NPs at 2 and 6 h were slightly less than 0.5% ID and did not differ significantly ( p>0.05) (Fig. 6B).
4. Discussion The results of the studies presented herein demonstrated: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of NPs with and without thiamine surface ligands, and (3) initial data suggesting mechanisms for NP brain entry. Comparison of the NP Kin demonstrated the [3H]thiamine-NPs associated with the BBB thiamine transporter and had increased brain uptake between 45 and 120 s. This data suggests that the thiamine ligand on the NPs may facilitate binding and/or association with BBB thiamine transporters. The relevance of this report is twofold; first, to our knowledge there is no other work that has specifically targeted NPs to brain in this manner, and second we have provided a highly sensitive kinetic analysis of NP movement at the BBB. The NPs used in this study were prepared using oil-in-water microemulsions as precursors. The ability to use microemulsions as precursors to engineer nanoparticles is in agreement with earlier studies [19]. The application of microemulsions as templates for NP production offers numerous advantages, demonstrated in this report, such as: (1) simplistic production of NPs ranging approximately 100 nm in diameter, (2) ease of microemulsion incorporation of hydrophobic compounds in the oil droplets (facilitating entrapment in cured NPs), and (3) inclusion of site-specific ligands in the NP preparations.
As shown in the TEM micrograph (Fig. 1), NP size averaged f 100 nm. The trend observed from laser light scattering also confirmed thiamine-coated NPs were f 100 nm with unimodal size distribution. Prior to brain perfusion studies, the stability of the NPs in relevant media was demonstrated (data not shown). Based on previous studies using liposomes and other macromolecules, we considered that the NP size of 100 nm as suitable for cell targeting and controlled release. The preparation technique also demonstrated significant versatility in that, for both the perfusion studies and biodistribution studies, different radiotracers were included in the NPs and entrapped at f 100% efficiency (data not shown) similar to previously published data [10]. The synthesized thiamine ligand has both DSPE groups and a PEG spacer. The DSPE hydrophobic groups were engineered to act as anchors and influence the insertion/adsorption of the thiamine ligand to the NP. The PEG spacer was included to facilitate flexibility and cell recognition of ligand on NP surface. Thiamine-coated NPs were obtained by adding 0.2% w/w thiamine ligand to NP suspensions at 25 jC. This NP formulation has been shown in-vitro to positively associate with human breast cancer cells expressing the thiamine transporters THTR1 and THTR2 [15]. To determine if the thiamine-coated NPs would similarly associate with BBB thiamine transporters we completed brain uptake evaluation for both the [3H]NPs and [3H]thiamine-NPs using the in-situ rat brain perfusion technique [11] (Fig. 2). The calculated transfer coefficient (Kin) based upon the initial 45 s linear uptake for the [3H]NPs is consistent with previously published in-situ NP perfusion data [10]. However, in contrast to the previous report, the [3H]thiamine-NPs had an initial brain distribution pattern that was non-linear in nature suggesting efflux or delayed brain penetration. Of interest, the placement of thiamine ligands on the NP created a brain uptake configuration consistent with [3H]thiamine brain uptake and efflux during insitu brain perfusions of less than 120 s [16]. Therefore, after evaluation of initial NP brain distribution we chose to evaluate [3H]thiamine-NP and [3H]NP efflux (Fig. 3) Specifically, the [3H]thiamine-NP complex contrasted [3H]thiamine BBB movement, in that there was no significant efflux during the washout
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study. However, similar to [3H]thiamine BBB movement there was no apparent BBB endothelial cell disassociation as seen by post-perfusion washouts resulting in an immediate reduction in the brain/ perfusion ratio [16,21]. Given the lack of efflux, we hypothesized the nonlinear [3H]thiamine-NP uptake pattern was secondary to: (1) facilitated but delayed transport into brain via the BBB thiamine transporter without subsequent efflux, and/or (2) the ligand –NP complex was associating with the carrier creating a subsequent delay in passive brain permeation. To determine if the NP was being transported across the BBB, experiments were completed to show substrate transporter association and saturability [20]. Therefore, we performed [3H]thiamine uptake experiments at 15 s with and without the presence of both NPs and unlabeled thiamine. The experimental time frame was based on previous [3H]thiamine BBB kinetic modeling (i.e. the time was long enough for [3H]thiamine brain detection yet was short enough to minimize apparent efflux) [16]. Fig. 4 shows addition of unlabeled thiamine (100 nM) to the perfusion fluid resulted in a reduction of PA consistent with previously published results [16]. Of major significance the uncoated NPs did not inhibit BBB [3H]thiamine transport yet the thiamine-NPs did (thiamine concentration f 100 nM). The ability of the latter NP type to inhibit BBB uptake [3H]thiamine strongly suggests NP complex association with thiamine BBB transporter proteins. To determine if the [3H]thiamine-NP was accessing brain via the BBB thiamine transporter, we evaluated the saturability of the process by the incorporation of unlabeled thiamine (50 AM) into the perfusion fluid. If transport occurs this concentration will completely inhibit BBB [3H]thiamine-NP brain distribution via the respective carrier [16]. In contrast to the expected finding (i.e. inhibiting brain penetration of [3H]thiamine-NP with unlabeled thiamine), Fig. 2 (inset) shows the presence of unlabeled thiamine resulted in a significant increase of [3H]thiamine-NP brain distribution. This data implies that addition of the thiamine ligand to the NP does not result in BBB penetration via facilitated thiamine BBB transport but rather the ligand – NP complex is associating with the thiamine BBB transporter with a subsequent delay in passive brain permeation.
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After determining the [3H]thiamine-NP was associating with the BBB thiamine transporter, we hypothesized there may be accumulation of the thiaminecoated NPs at the BBB. Potentially, NP accumulation at the BBB may lead to improved brain uptake by increasing the concentration gradient. To assess this hypothesis, we extended perfusion time frames to 60, 75, 90 and 120 s. In contrast to the non-linear and linear brain uptake pattern seen in the initial 45 s time frame, both NPs had an apparent linear brain uptake profile during the extended perfusions. Fig. 5 shows the uptake transfer coefficient (Kin) for [3H]thiamineNPs is significantly greater than the [3H]NPs during the perfusion 45 to 120 s time frame. This data further supports the hypothesis of the thiamine ligand associating with the thiamine BBB transporter may improve overall NP brain uptake. To assess the improved NP brain uptake we completed biodistribution studies. The NPs were modified to contain a DSPE-PEG spacer based upon: (1) retention of the cured NPs in blood [24] and (2) PEG-coated macromolecules display specific affinity for brain endothelial cells [25]. As shown in Fig. 6A, both thiamine and PEG-coated (control) NPs had long circulating properties most likely due to PEG molecules preventing or at least minimizing reticuloendothelial NP recognition. It is also noteworthy there was an apparent lack of NP aggregation (leading to rapid lung deposition) as demonstrated by the low levels of radioactivity observed in the lungs (Fig. 6A). For both thiamine-coated and PEG-coated NPs, low levels of radioactivity were distributed to the brain at the time points studied. In the biodistribution studies (Fig. 6B), thiamine ligand coating on NPs did not have a significant effect on brain radioactivity levels (in comparison to control) possibly due to a number of factors including: (1) addition of 0.2% w/w of thiamine ligand may result in low and insufficient density of thiamine ligand coating on NPs in-vivo, (2) brain distribution at extended time points (beyond 6 h) were not studied, (3) normal blood thiamine levels in mice may competitively inhibit brain uptake of thiamine-coated NPs, (4) the thiamine NP complex may interact with erythrocyte thiamine transporters and (5) in-vivo shear flow and dilution effects could diminish the amount of thiamine-coated NP association with BBB.
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In summary, the results presented herein indicate the addition of a thiamine ligand to the NPs causes association with the BBB thiamine transporter. This association may create an accumulation of NPs at the BBB, which ultimately increases brain uptake during perfusion time frames. Of major importance, the blood retention properties of both NPs may increase their potential utility in site-specific and controlled drug delivery.
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