2018 年 41 巻 4 号 p. 546-554
To overcome the difficulty in delivery of biopharmaceuticals such as peptides and proteins to the brain, several approaches combining the ligands and antibodies targeting the blood–brain barrier (BBB) have been tried. However, these are inefficient in terms of their permeability through the BBB and structural modification of bioactive drugs. In the present study, we therefore examined the usefulness of a noncovalent method using the cell-penetrating peptides (CPPs) such as octaarginine (R8) as a suitable brain delivery strategy for biopharmaceuticals. A safety examination using microvascular endothelial model bEnd.3 cells clarified that R8 was the safest among the CPPs tested in this study. The cellular uptake study demonstrated that coincubation with R8 enhanced the uptake of model peptide drug insulin by bEnd.3 cells in a concentration-dependent and a temperature-independent manner. Furthermore, an in vivo study with rats showed that the accumulation of insulin in the deeper region of the brain, i.e., hippocampus, significantly increased after the intravenous coadministration of insulin with D-R8 without altering the insulin disposition in plasma. Thus, the present study provided the first evidence suggesting that the noncovalent method with CPPs is one of the strategic options for brain delivery of biopharmaceuticals via intravenous injection.
Brain is the most critical organ that governs functions such as learning, memory, and emotion, and is intimately connected to the functions of other organs in the body. Disorders and impairment in brain functions severely complicate the lives of patients and their family. Therefore, the brain is a sanctuary strictly protected from the external environment. In particular, the blood–brain barrier (BBB), formed by the microvascular endothelial cells, pericytes, and astrocytes, prevents the influx of exogenous substances from the systemic blood to the brain.1) While BBB has an essential role in protecting the brain, it poses a challenge by extremely limiting the delivery of therapeutic agents in the treatment of central nervous system (CNS) diseases. While the hydrophilic molecules cannot pass through the narrow paracellular spaces of the endothelium, a majority of the hydrophobic molecules including anticancer drugs are recognized as substrates for the efflux transporters such as P-glycoprotein and breast cancer resistance protein.2–6) Furthermore, the transport of macromolecular bioactive peptides and proteins is strictly limited via both paracellular and transcellular pathways.
Recent advances in the pharmacological research field strongly suggested the potentials of various peptide- and protein-based biopharmaceuticals such as insulin, oxytocin, neurotrophins and antibody drugs for the treatment of dementia, autism and others.7–10) Among them, the antidiabetic peptide drug insulin is expected to have the potential to cure forms of dementia such as Alzheimer’s disease,7,8,11) and it is currently considered as an agent to accelerate the cognition and learning by stimulating its receptor in the brain and by inhibiting the cytotoxic action of amyloid β oligomer.12) However, as mentioned above, these biological agents cannot reach at the brain parenchyma by the hindrance of BBB. Thus, a strategy for drug delivery to the brain must be developed as an effective pharmacotherapy for the CNS diseases.
The difficulties in drug delivery to the brain include low accumulation of drugs onto the BBB from systemic circulation and their poor permeability through the BBB. Over the decades, several challenges have been overcome to facilitate drug transport through brain endothelial cells. For instance, receptor-mediated transcytosis has been used as one of the typical strategies.13–21) However, this method requires covalent conjugation of the drugs with ligands for transferrin receptor and low density lipoprotein-related protein-1 (LRP1), requiring complicated preparation procedures and therefore raising concerns on the loss of therapeutic activity because of structural modifications. Another typical strategy that uses tight junction opening agents might be linked to the nonspecific influx of undesirable pathogens.3,22,23)
In the context of above-mentioned situation, we have successfully developed a noncovalent strategy using cell-penetrating peptides (CPPs) to enhance the permeation of biopharmaceuticals such as peptide- and protein-drugs, through biological membranes.24–27) The CPPs are useful tools for intracellular delivery of macromolecular drugs, and generally they are being used in drug delivery systems via covalent chemical conjugation.28–30) In contrast to these conventional approaches, we found that the systemic absorption of biopharmaceuticals through intestinal and nasal epithelium dramatically increased by noncovalent coadministration with CPPs.24–27) In the noncovalent strategy, it was demonstrated that the peptide drug molecules make soluble complex with CPPs via electrostatic and/or hydrophobic force, and the molecular complex is a key factor for governing absorption enhancement effect of CPPs.25) While CPPs tended to accumulate in the cell fraction, the active peptide drugs can be dissociated from the molecular complex after their cellular internalization and eventually permeate through the basement membrane of the epithelium.27) Furthermore, we recently found that noncovalent coadministration with CPPs can facilitate the transport of peptide drug insulin from nasal cavity to the brain.31,32)
The noncovalent strategy with CPPs, in addition to the above-mentioned effective function, it has another merit that the noncovalent physical mixture of bioactive drugs and CPPs is easy to prepare, and there is no possible loss of pharmacological activity through structural modifications. Thus, noncovalent strategy with CPPs is regarded as effective and practical delivery tool for accelerating the epithelial uptake of biopharmaceuticals, whereas the noncovalent method has been untested so far for the drug delivery through BBB following intravenous injection. Therefore, in this study, we examined the usefulness of a noncovalent strategy with CPPs to accelerate the brain endothelial transport of biopharmaceuticals such as insulin following their intravenous systemic administration and eventual delivery to the brain.
Recombinant human insulin (27.5 IU/mg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The CPPs listed in Table 1 were synthesized by Sigma-Genosys, Life Science Division of Sigma-Aldrich Japan Co. (Hokkaido, Japan). Cy7-NHS ester was purchased from GE Healthcare UK Ltd. (Buckinghamshire, U.K.). Fluorescein isothiocyanate (FITC)-labeled dextran with average molecular weight 4400 (FD-4), p-chloromercuribenzoic acid (PCMB) was purchased from Sigma-Aldrich Co. (Darmstadt, Germany). Protease inhibitors cocktail tablet (cOmplete) was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Mouse brain endothelioma-derived bEnd.3 cells were purchased from American Type Culture Collection (Manassas, VA, U.S.A.) at passage 22. Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, nonessential amino acids (NEAA), antibiotics mixture (10000 U/mL penicillin, 10 mg/mL streptomycin, and 29.2 mg/mL of L-glutamine in 10 mM citric acid-buffered saline), 0.05% trypsin–ethylenediaminetetraacetic acid (EDTA), and Hank’s balanced salt solution (HBSS) were purchased from Gibco Laboratories (Lenexa, KS, U.S.A.). Fetal bovine serum (FBS) was purchased from Biowest (Nuaille, France). RIPA buffer was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, U.S.A.). All other chemicals were of analytical grade and are commercially available.
| Peptides | Sequencesa, b) |
|---|---|
| L-R8 | RRRRRRRR |
| D-R8 | rrrrrrrr |
| L-Tat | GRKKRRQRRRPPQ |
| D-Tat | grkkrrqrrrppq |
| L-Penetratin | RQIKIWFQNRRMKWKK |
| D-Penetratin | rqikiwfqnrrmkwkk |
a) F: phenylalanine, G: glycine, I: isoleucine, K: lysine, M: methionine, N: asparagine, P: proline, Q: glutamine, R: arginine, W: tryptophan. b) Uppercase and lowercase letters indicate the L- and D-forms of the amino acids, respectively.
Specific amounts of CPPs (R8, Tat, and penetratin, as listed in Table 1) were dissolved in HBSS or phosphate-buffered saline (PBS, pH 7.4) containing 0.001% methylcellulose, which prevents adsorption of CPPs to the tube wall. For cell studies, FD-4 and insulin were dissolved in HBSS with 0.001% methylcellulose to prepare 7.0 mg/mL and 150 µM of stock solutions, respectively, and then, the stock FD-4 or insulin and the CPP solutions in HBSS were added separately to the cells to obtain their final concentrations described in the “Cellular uptake study section.” For animal studies, a specific amount of insulin was dissolved in PBS (pH 7.4) with 0.001% methylcellulose and then mixed with an equal volume of D-R8 solution (4 mM in PBS), just before being administered to the rats.
Labeling of Insulin with Cy7 Fluorescent DyeSpecific amount of insulin was dissolved in PBS (pH 7.4) with methylcellulose, and then 2 mL of the insulin solution (5 mg/mL) was desalted, using PD-10 column (GE Healthcare). Insulin was eluted with 50 mM NaHCO3 solution and the fraction of eluted solution (3.0–6.0 mL) was collected (3.33 mg/mL of insulin). Cy7-NHS ester (1 mg) was dissolved in 0.125 mL of dimethyl sulfoxide and added in 1.8 mL of the eluted insulin solution. After the addition of 15 µL of trimethylamine to the Cy7-ester and insulin solution, the mixed solution was gently shaken overnight in the dark at room temperature. The reacted solution (1.94 mL) was desalted by using a PD-10 column, and then eluted with PBS (pH 7.4) with 0.001% methylcellulose. The fraction of eluted solution (4.0–6.0 mL) was collected, and quantified using HPLC (LaChrom Elite system, Hitachi High-Technologies Corp., Tokyo, Japan) using following conditions for mobile phase (acetonitrile–trifluoroacetic acid (0.1%): sodium chloride (31 : 69 : 0.58, v/v/w)), injection volume (20 µL), flow rate (1.0 mL/min), wavelength (220 nm), column 4.6×150 mm, 5 µm (GL-Pack Nucleosil 100-5C18, GL Science Inc., Tokyo, Japan). The concentration of Cy7-labeled insulin was 0.53 mg/mL (equivalent to approximately 15 IU/mL). The diluted Cy7-insulin (0.3 IU/mL) was mixed with equal volume of D-R8 solution (4 mM) in PBS just before the biodistribution study.
Cell CulturebEnd.3 cells were cultured in 75 cm2 culture dishes (Becton Dickinson, Franklin Lakes, NJ, U.S.A.) with 10 mL of culture medium comprising DMEM with 10% FBS, 0.1 mM NEAA, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The seeding density for culture was 8.0±105 cells/dish. The cells were maintained in an incubator at 37°C, 95% relative humidity, and 5% CO2. When the cells reached 80% confluence, they were subcultured. In the subculture procedure, the cells were detached from the culture dishes by trypsinization with 0.05% trypsin–EDTA, counted with a hemocytometer, and transferred at the desired seeding density to new culture dishes or experimental wells. The cells used in these studies were between passages 25–35.
Cellular Uptake StudybEnd.3 cells were grown at a density of 2.0±105 cells/cm2 in 24-well Multiwell plates with a 1.88 cm2 culture area (Becton Dickinson). The cells were grown in a DMEM-based culture medium for 7 d. The culture medium was changed every other day. Before initiation of the uptake experiments, the cell membrane was allowed to equilibrate for 30 min in 0.5 mL of transport buffer at 37 or 4°C. HBSS (pH 7.4) containing 0.001% methylcellulose was added to the wells as the transport buffer. After a 30 min preincubation with the transport buffer, the experiment was initiated by replacing 100 µL transport buffer with 50 µL of stock FD-4 (7.0 mg/mL) or insulin (150 µM) solution and 50 µL of CPP solution (50–2400 µM) or the transport buffer alone. A solution of FD-4 (0.7 mg/mL) or insulin (15 µM) was incubated independently or simultaneously with CPPs (5–240 µM) in the wells seeded with bEnd.3 cells at biological (37°C) or energy-abolishing (4°C) conditions. After the designated period, the cells were washed seven times with 1 mL of ice-cold HBSS to remove the surface-bound dextran, insulin, and CPPs (similarly effective way to wash with heparin solution), and then solubilized by the addition of 300 µL of ice-cold RIPA buffer to the wells. In the study with insulin, the insulin-degrading enzyme inhibitor, PCMB, was added in RIPA buffer at 0.2 mM. The cell lysates were then centrifuged at 4°C and 14000×g for 15 min, and the concentrations of FD-4 in the supernatants were measured with a microplate fluorometer (Synergy HT, BioTek Instruments Inc., Winooski, VT, U.S.A.) at excitation and emission wavelengths of 485 and 528 nm, respectively. The insulin concentrations in the supernatants were determined using an enzyme-linked immunosorbent assay (ELISA)-based human insulin assay kit (Mercodia AB, Uppsala, Sweden). The residual aliquots of supernatants were used to determine the protein concentrations using BCA assay reagents (Thermo Fisher Scientific Inc.) with bovine serum albumin as the standard protein. The uptake of FD-4 and insulin by bEnd.3 cells at each time point was calculated by dividing the uptake amount (corrected by the cellular protein content) by the initial concentration.
Cytotoxicity AssayTo assess the cytotoxic effects of the CPPs, the integrity of the cell membrane after treatment of the CPPs, with or without FD-4 or insulin, was examined by determining the lactate dehydrogenase (LDH) released from the cytoplasm. In these assays, the bEnd.3 cells were grown in 24-well Multiwell plates, as described in the cellular uptake study. After 30 min of preincubation with transport buffer (HBSS with 0.001% methylcellulose), various concentrations (150, 300, 600 and 1200 µM) of L- or D-form of CPP were incubated with or without FD-4 (0.7 mg/mL) or insulin (15 µM) in bEnd.3 cell-seeded Multiwells for 120 min at 37°C. The incubation buffer was then collected, and the LDH released from the cytoplasm into the incubation buffer was determined using a CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega Corp., Madison, WI, U.S.A.). Cytotoxicity was expressed as the percentage calculated by dividing the absorbance of the vehicle (HBSS) or the CPP-treated sample by that of a sample treated with 0.8% Triton X-100.
In Vivo Intravenous Administration StudyThe animal study was performed at Kobe Gakuin University in compliance with the regulations of the Committee on Ethics in the Care and Use of Laboratory Animals of that institution. Male Sprague-Dawley rats (180–220 g) were purchased from Japan SLC Inc. (Shizuoka, Japan). All rats were housed in rooms maintained under a 12 h light–dark cycle at 23±1°C and 55±5% relative humidity, and were allowed free access to water and food during acclimatization. For the ex vivo imaging study, rats were fed non-fluorescent rodent diet (D10001, Research Diets Inc., New Brunswick, NJ, U.S.A.) for a week before intravenous administration to avoid an intrinsic fluorescence of tissues.
Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Somnopentyl, Kyoritsu Seiyaku Corp., Tokyo, Japan), and they were restrained in a supine position. A 200 µL of insulin (0.5 IU/mL) or Cy7-insulin (0.15 IU/mL) solution with or without D-R8 (2 mM) was intravenously administered via the left jugular vein. The doses of insulin and Cy7-insulin were 0.5 and 0.15 IU/kg body weight, respectively.
Measurement of Insulin Concentrations in Plasma and Brain SamplesSeven blood samples (200 µL) from each rat were collected from the right jugular vein using 1 mL tuberculin heparinized syringes (Terumo Corp., Tokyo, Japan) at 0, 2, 5, 10, 15, 30, and 60 min after intravenous administration of insulin (0.5 IU/kg) with or without D-R8 (2 mM). After blood sampling at 60 min, the abdominal cavity was opened, and the intravascular content in the brain was flushed by perfusing ice-cold PBS (pH 7.4) into the left ventricle of the heart at 15 mL/min for 5 min using a peristaltic pump (ATTO Corp., Tokyo, Japan). The rat was decapitated, and the whole brain was carefully isolated and washed with ice-cold PBS. The isolated brain sample was separated into six parts—olfactory bulbs, hypothalamus, hippocampus, cerebral cortex, cerebellum, and brainstem. These samples were weighed and homogenized with a twice volume of ice-cold 2-(N-morpholino)ethanesulfonic acid (MES) solution (pH 3.5) with a protease inhibitor cocktail using a glass or microtube tissue grinder. The blood samples and homogenized samples were centrifuged at 4°C and 5400×g for 15 min, and the insulin concentration in the resultant plasma or homogenate supernatant was measured using an ultrasensitive insulin ELISA kit (Mercodia AB). In this study, the supernatant of brain homogenate obtained following above method was used for determining the concentration in the brain parenchyma, although the capillary contents were not depleted.
Biodistribution Study Based on ex Vivo ImagingThe biodistribution of insulin after its intravenous administration with or without D-R8 was analyzed by using in vivo imaging system (IVIS LuminaXR, Caliper Life Sciences, Inc., Waltham, MA, U.S.A.). To detect the fluorescent signal in the deeper organs, the organs were analylzed at ex vivo condition. At the designated time point (15 min) after intraveous administration of Cy7-insulin (0.15 IU/kg) with or without D-R8 (2 mM), the rat was decapitated, and the organs including the brain, kidney, lung, spleen, heart, liver and duodenum were carefully collected. The isolated organs were washed with ice-cold PBS, and mounted on the stage of in vivo imaging system. The distribution characteristics of fluorescence derived from Cy7 to the organs were analyzed at excitation and emission wavelengths of 747 and 776 nm, respectively. The fluorescent intensity of the region of interest (ROI) of image was semi-quantified based on their contrast. The fluorescent intensity of organs isolated from the rats receiving Cy7-insulin with or without D-R8 was calculated by subtracting the intrinsic fluorescence of untreated rats from their total intensity.
Statistical AnalysisEach value was expressed as the mean and standard error of the mean (S.E.M.) of multiple determinations. The significance of differences in the mean values of two groups was evaluated using Student’s unpaired t-test. For multiple comparisons with the control group, ANOVA with Dunnett’s test was applied. IBM SPSS Statistics Version 23 (IBM Corp., Armonk, NY, U.S.A.) was used for statistical analysis. Differences were considered significant at a p value <0.05.
In this study, we selected three major CPPs—octaarginine (R8), Tat peptide, and penetratin (Table 1)—as the potential CPPs for enhancing the permeation of macromolecular drugs across the BBB, and examined firstly the safety in the use of these CPPs by measuring their cytotoxicity to the microvascular endothelial model bEnd.3 cells. Figures 1A and B shows the release of the intracellular protein—LDH—from bEnd.3 cells after the addition of various concentrations of the CPPs. Among the three CPPs that were tested, a slight release of LDH (approximately 10%) was detected in the media (HBSS), at higher concentration (120 µM) of L- or D-penetratin. In contrast, the level of LDH released after exposure to R8 and Tat was similar (less than 5%) to that in the blank condition (0 µM of CPPs), at all the tested concentrations. In addition, the coincubation of CPPs with other macromolecules such as hydrophilic model compound (FD-4) and insulin did not affect the safety of CPPs, as shown in Figs. 1C and D.
The LDH concentrations in the samples collected at 120 min after exposure with CPPs (L- or D-form of R8, Tat, or penetratin) in the absence (panels A and B) or presence of FD-4 or insulin (panels C and D). Panels A and C show the results with L-CPPs, and panels B and D show that with D-CPPs. Each data represents the mean±S.E.M. of n=3–4. * p<0.05, significant differences compared to “0 μM” control.
As described above, the L- and D-forms of R8 and Tat peptide were confirmed to be safe to the brain microvascular endothelium. Therefore, the effect of these two CPPs on the endothelial uptake of macromolecules was examined using bEnd.3 cells. Figure 2A shows the time-dependent uptake profiles of FD-4, a model hydrophilic macromolecule. The result demonstrated that the uptake of FD-4 by the bEnd.3 cells significantly increased after its coincubation with D-R8 (60 µM). In contrast, no enhancement was detected in FD-4 uptake by the bEnd.3 cells after cotreatment with D-Tat (60 µM). Further, the L- and D-forms of Tat and R8 were added to the peptide-drug insulin, and their effect on the cellular uptake of insulin was tested. As shown in Figs. 2B and C, all of four CPPs tended to enhance the uptake of insulin by the bEnd.3 cells. In particular, the effect of R8 (both L- and D-forms) was stronger than that of Tat, and both L- and D-R8 stimulated the uptake of insulin in equal capacities. Based on the result shown in Fig. 2, we further examined the concentration-dependent action of L- and D-forms of R8. As shown in Fig. 3A, the uptake of insulin by bEnd.3 cells increased after coincubation with L- or D-R8, in a dose-dependent manner. In particular, an approximately 10-fold increase in insulin uptake was detected after coincubating with 240 µM of L- and D-R8 (i.e., 5.4 and 5.5 µL/mg protein/30 min with 240 µM of L- and D-R8, respectively vs. 0.54 µL/mg protein/30 min at 0 µM CPPs.). This suggested that L- and D-R8 had a stronger ability to enhance the cellular uptake of insulin and other macromolecular drugs into the brain microvascular endothelium.
The bEnd.3 cells were treated with FD-4 (A) or insulin (B and C) in the absence or presence of CPPs (60 µM). FD-4 was added with D-R8 and D-Tat (A), and insulin was added with L- and D-Tat (B) or L- and D-R8 (C). Each data point represents the mean±S.E.M. of n=3–20. * p<0.05, significant differences compared to corresponding control “FD-4” or “Insulin” without CPPs.
Several workers have suggested that endocytosis such as macropinocytosis might be associated with the effective internalization of CPPs into the cells.33–35) Therefore, we tested the involvement of energy-dependent internalization mechanisms in the action of CPPs on the bEnd.3 cells by comparing the uptake studies under biological (37°C) as well as under energy-abolishing (4°C) conditions. The result, shown in Fig. 3B, demonstrated that the uptake of insulin by bEnd.3 cells was significantly decreased at 4°C, both in the presence and absence of D-R8 (120 µM). Meanwhile, the stimulatory effect (6.1-fold increase at both temperatures) of D-R8 (120 µM) on the cellular insulin-uptake was notably maintained at 4°C. These data suggest that although the uptake of insulin itself by the bEnd.3 cells changed at different temperatures, the action of D-R8 on the insulin uptake might be mediated in an energy-independent manner.
Panel A: bEnd.3 cells were treated with insulin (15 µM) and various concentrations (30–240 µM) of L- or D-R8 at 37°C. Panel B: bEnd.3 cells were treated with insulin and D-R8 (120 µM) at 37 or 4°C. Each data point represents the mean±S.E.M. of n=3–10. * p<0.05, ** p<0.01, significant differences in insulin uptake compared to “0 μM of R8” at the same temperature. ## p<0.01, significant differences in insulin uptake at same application groups between different temperatures.
As described above, it was demonstrated that L- and D-R8 could safely and effectively increase the macromolecular drug uptake, particularly that of insulin, under the in vitro condition, emphasizing on the possibility of use of noncovalent CPP strategy for brain delivery of such drugs. Therefore, we tried to show the in vivo potential of CPP coadministration for brain delivery of insulin. Figure 4A shows the time profiles of plasma insulin concentrations after intravenous bolus injection of insulin with or without D-R8 (2 mM), where only the unnatural D-form of R8 was selected because of its higher stability than L-R8 as shown in our previous work.36) No significant change in plasma insulin concentration was observed after coadministration with D-R8, at all the time points, suggesting that the clearance of the systemically injected insulin was not affected by the coadministered D-R8. After blood sampling at 60 min post-injection, the distribution of insulin in six parts of the brain was measured. The absolute amount of insulin that can reach at the brain was still quite low even when enhanced by coadministration with D-R8, therefore the concentration of insulin in the brain was determined by using ultrasensitive ELISA kit (detection range: 0.15–20 μU/mL), which has 10 times sensitive compared to normal version of ELISA kit (detection range: 3–200 μU/mL). Overall, except for the olfactory bulb, the concentration of insulin in the brain tended to increase after coadministration with D-R8 than that without D-R8 (Fig. 4B). Especially, the hippocampal insulin concentration was statistically higher following coadministration with D-R8. These data suggested that a noncovalent strategy using CPPs can facilitate the influx of insulin into brain parenchyma without affecting the systemic circulation of insulin.
Panel A shows the time profile of plasma insulin concentration after intravenous injection of insulin (0.5 IU/kg) with or without D-R8 (2 mM) for 60 min. Panel B shows the insulin concentration in six parts of the brain at 60 min after intravenous injection of insulin with or without D-R8 (2 mM). Each data point represents the mean±S.E.M. of n=3–6. * p<0.05, significant differences in insulin concentration compared to the “Insulin” (control).
Finally, we confirmed the biodistribution of Cy7-labeled insulin after its intravenous injection with D-R8 based on the ex vivo fluorescent imaging. Since our previous works demonstrated that CPPs tended to accumulate inside of cells and could not permeate through the cell layer, we did not analyze D-R8 transport by fluorescently labeling D-R8 molecules in this study.27,37) Figures 5B and C shows the typical images and the detected fluorescence from seven organs isolated from the rats that received the Cy7-insulin injection with and without D-R8, respectively (Fig. 5A, untreated rat as the blank). In addition, Fig. 5D shows the fluorescent intensity in the organs, calculated based on the contrast, reflecting the distribution levels. The results demonstrated that hepatic and renal distributions of Cy7-insulin significantly increased after coadministration with D-R8, emphasizing the ability of D-R8 to deliver the drugs throughout the body without targeting to specific parts. In this ex vivo imaging assay, the fluorescence of Cy7-insulin could not be clearly detected in other organs including brain, because of its insufficient sensitivity for quantification.
Panels A–C show the typical images obtained from the rats without treatment (A) and receiving the intravenous injection of Cy7-insulin (0.15 IU/kg) without (A) or with D-R8 (2 mM) (C). Panel D shows the fluorescent intensity in the tissues calculated based on the contrast in images obtained from multiple experiments. Each data point represents the mean±S.E.M. of n=3–4. * p<0.05, significant differences in fluorescent intensity compared to “Cy7-Insulin” (control). N.D., Not detected.
Recent advances in neurobiology and neuropathology motivated us to use different peptides and proteins as novel drug candidates for treating CNS disorders, such as dementia, autism, and metabolic syndromes.7–10) However, the BBB is a major and strict hurdle for the delivery of biopharmaceuticals to the brain.1) Therefore, there is a need to establish a reliable strategy to facilitate the permeability of such drugs across the BBB. Our recent works suggested that use of CPPs in noncovalent form could be an ideal strategy for the delivery of biopharmaceuticals over biological membrane and eventually for the transcellular permeation barriers such as the intestinal and nasal epithelium.24–27) In this study, we therefore tried to expand our strategy with CPPs for drug delivery to the brain, the most difficult target site in the body.
Before testing the usefulness of CPPs as a tool for brain drug delivery, their safety to the brain microvascular endothelial cells that form the interface between the blood and the brain was preliminarily tested. For cytotoxicity examination using bEnd.3 cells, we selected three typically used CPPs—R8,38,39) Tat,40) and penetratin41) — because we had previously found that they could significantly enhance the absorption of peptides and proteins through the intestinal and nasal epithelium.24–26) The results suggested that L- and D-forms of R8 and Tat were quite safe for the bEnd.3 cells, at all of the tested concentrations (Fig. 1). However, only penetratin showed a slight increase in LDH leakage from bEnd.3 cells, although, in our previous studies, this functional peptide was shown to be safe to intestinal epithelial model Caco-2 cells.27) Therefore, we were obliged to exclude penetratin in following examinations for safe use in drug delivery to the brain.
The in vitro cellular uptake study suggested that R8 and Tat had the potential to deliver hydrophilic macromolecules such as dextrans and insulin into bEnd.3 cells (Figs. 2A–C). The stimulatory effect of R8 on the cellular insulin uptake was strongest around 30 min after the addition of CPPs to the media (Fig. 2C). Probably, these stimulation profiles of insulin uptake were derived from a balance between the cellular uptake and the degradation in the cells, equivalent to that of our previous study with Caco-2 cells.27) The concentration-dependent study using the L- and D-forms of R8 unexpectedly demonstrated that the unstable L-form of R8 had a strong stimulatory effect on the insulin uptake in a concentration-dependent manner similar to that of the D-R8 form (Fig. 3A). The residual effect of L-R8 suggested that L-R8 might be less sensitive to degradation in the cell-exposed media and in the cytoplasm of bEnd.3 cells, which was contrary to our previous study using Caco-2 cells that probably provided a harsher degradation environment.27) On the other hand, many reports have suggested that CPPs are internalized into numerous cells via both endocytosis such as macropinocytosis and energy-independent direct translocation.33–35,42–46) The stimulatory effect of D-R8 on the insulin uptake by bEnd.3 cells was completely maintained at the energy-abolishing condition (4°C), although the intrinsic uptake of insulin, probably via insulin receptor-mediated endocytosis, decreased at low temperature (Fig. 3B). This corresponded to our previous study,27) suggesting that direct translocation might be involved in the internalization of D-R8 and its stimulatory effect on the insulin uptake. Some reports suggested the involvement of both endocytosis and direct translocation in the internalization of CPPs into the cells, however, the detailed mechanisms remained unclear.
The favorable results obtained from in vitro study using bEnd.3 cells were encouraging and allowed our strategy with CPPs to proceed to the next examination stage. In the in vivo examination, D-R8 was selected as the most effective, safe and enzymatically stable CPP for the drug delivery via intravenous administration, and was coadministered at a higher concentration (2 mM), taking into account the dilution after systemic administration. Totally, the concentration of insulin in the brain tended to increase after the intravenous coadministration of D-R8, in the rats (Fig. 4B). It is noteworthy that the increase in hippocampal insulin concentration was significant, although the reason for the increased distribution of insulin into such deeper regions of the brain remained unclear. The highly expressed insulin receptor might be involved in the effective transport of insulin to hippocampus, as well as its contribution to the activation of insulin signaling that is linked to the memory, cognition and learning.47) The effect of D-R8 administered as a noncovalent form in the physical mixture was significantly meaningful, since the conventional methods that use specific ligands such as transferrin and Angiopep-2 usually require covalent linkage to drugs.13,15,19–21) Only one study suggested that synthetic carrier peptide combining sixteen lysine residues and low-density lipoprotein receptor-binding sequence (K16ApoE) had the potential to deliver the immunoglobulins to brain via noncovalent manner.48) However, the delivery of immunoglobulins into the brain as well as other organs was achieved by larger carrier peptide with total 36 amino acids, and its finding was obtained by the semi-quantitative analysis without cytotoxicity examination; therefore, this cannot exclude the toxic possibility of K16ApoE peptide to the endothelial membranes.
As described above and shown in our previous publications,25,27) the intermolecular interaction between drugs and CPPs via electrostatic and/or hydrophobic forces is essential for noncovalent form of CPPs to enhance the strategy. When the blood volume of rat (200 g) is hypothesized to be 10 mL, the administered solution (200 µL) including insulin (0.5 IU/mL=3.13 µM) and D-R8 (2 mM) is calculatedly diluted 50 times after intravenous administration, and eventually the concentration in systemic circulation might be 62.6 nM and 40 µM, respectively. Then, the concentration of insulin and D-R8 as the bound forms could be calculated to 50.7 nM (81% of total insulin) and 95 nM (0.24% of total D-R8), respectively, by using the binding parameters, dissociation constant (Kd) and maximum binding capacity (Bmax) obtained in our previous study.25) Based on the calculation, a large portion of insulin is considered to be present as the complex form with D-R8 in the blood, which can contribute to the enhanced permeation through BBB.
Unfortunately, the ex vivo imaging could not detect the distribution of fluorescence-labeled insulin in the brain due to low sensitivity in this assay. However, the distribution of insulin to the clearance organs such as liver and kidney by coadministration of D-R8 was clearly observed (Fig. 5). This means the low targeting ability of the CPPs such as D-R8 to specific organs. The increased hepatic and renal uptake of insulin might counteract the protective effect on systemic degradation of insulin, and eventually no change was detected in the pharmacokinetic disposition of insulin in blood (Fig. 4A).
In conclusions, this is the first study demonstrating that the noncovalent method with CPPs is one of the options that can be utilized in brain delivery strategy of biopharmaceuticals via intravenous administration. However, combination of the brain-targeting agents or carriers that can concentrate the drugs and CPPs onto the brain endothelial surface would be needed to further increase the usefulness of CPPs for drug delivery to the brain via systemic administration.
This study was supported in part by a research Grant from the Hyogo Science and Technology Association and the Kobe Gakuin University Research Grant A.
The authors declare no conflict of interest.
