Stability and Structure of ProteinâLipoamino Acid Colloidal Particles

Article pubs.acs.org/Langmuir

Stability and Structure of Protein−Lipoamino Acid Colloidal Particles: Toward Nasal Delivery of Pharmaceutically Active Proteins Christian Bijani,†,‡ Clément Arnarez,† Sabrina Brasselet,‡ Corinne Degert,‡ Olivier Broussaud,‡ Juan Elezgaray,† and Erick J. Dufourc*,† †

Institute of Chemistry & Biology of Membranes & Nanoobjects, UMR 5248, CNRS, Université Bordeaux, Institut Polytechnique Bordeaux, Pessac, France ‡ Physica Pharma, Pessac France ABSTRACT: To circumvent the painful intravenous injection of proteins in the treatment of children with growth deficiency, anemia, and calcium insufficiency, we investigated the stability and structure of protein−lipoamino acid complexes that could be nasally sprayed. Preparations that ensure a colloidal and structural stability of recombinant human growth hormone (rhGH), recombinant human erythropoietin (rhEPO), and salmon calcitonin (sCT) mixed with lauroyl proline (LP) were established. Protein structure was controlled by circular dichroism, and very small sizes of ca. 5 nm were determined by dynamic light scattering. The colloidal preparations could be sprayed with a droplet size of 20−30 μm. The molecular structure of aggregates was investigated by all-atom molecular dynamics. Whereas a lauroyl proline capping of globular proteins rhGH and rhEPO with preservation of their active structure was observed, a mixed micelle of sCT and lipoamino acids was formed. In the latter, aggregated LP constitutes the inner core and the surface is covered with calcitonins that acquire a marked αhelix character. Hydrophobic/philic interaction balance between proteins and LP drives the particles' stability. Passage through nasal cells grown at confluence was markedly increased by the colloidal preparations and could reach a 20 times increase in the case of EPO. Biological implications of such colloidal preparations are discussed in terms of furtiveness.

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INTRODUCTION Most of the protein medicinal drugs are clinically administered through an intravenous injection several times a week. The repeated injections cause pain to patients; hence, an alternative route of administration is desirable such as oral or nasal routes. Unfortunately, proteins are rapidly destroyed by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via the oral route.1 We explored in this work the nasal route by formulating three proteins as colloidal suspensions, in such a way that they could be administered as a spray solution. Human growth hormone (hGH) (Figure 1A), a single polypeptide chain of 191 amino acids and a molecular mass of 22 kDa, is a somatropic hormone secreted from the anterior pituitary gland. The release of hGH is regulated by the growth hormone releasing hormone (GHRH) secreted from the hypothalamus2 in an episodic and pulsatile manner.3 The recombinant human growth hormone (rhGH) is used in the treatment for growth hormone-deficient children with short stature. Calcitonin, which is a polypeptide of 32 amino acids with a molecular mass of 2.5 kDa, has a physiological role in the regulation of calcium homeostasis and is a potent inhibitor of osteoclastic bone resorption.4 Salmon calcitonin (sCT) (Figure 1B) is widely used because it is the most potent of the calcitonins available, it is well tolerated, and it is clinically effective.5 Erythropoietin (EPO) is a glycoprotein of 166 amino © 2012 American Chemical Society

acids with a molecular weight of 34 kDa. EPO is produced mainly in the kidney and stimulates proliferation and differentiation of erythroid precursor cells to red blood cells. Recombinant human erythropoietin (rhEPO) (Figure 1C) has been widely used clinically as an effective treatment of anemic patients with insufficient EPO production, for example, in endstage renal disease. rhEPO is also approved for anemic patients suffering from neoplastic disease and who acquired immunodeficiency with azidhothymidine treatment.6 The intranasal approach is not common for the delivery of proteins, but there are some reports on its use for the administration of rhGH. For instance, the effect of the bile salt derivative sodium tauro-24,25-dihydrofusidate (STDHF) on the nasal absorption of rhGH in the rat, rabbit, and sheep has been evaluated.7 Compared to a single aqueous solution of rhGH, the addition of STDHF produced a 11-fold increase in intranasal rhGH bioavailability in rats and rabbits and a 21-fold increase in sheep. STDHF-based rhGH solution was also administered to growth-hormone-deficient patients.8 The bioavailability relative to subcutaneous injections is usually very low, in the range 1−3%, but interestingly, preparations containing rhGH and didecanoyl-L-α-phosphatidylcholine have Received: January 17, 2012 Revised: March 5, 2012 Published: March 6, 2012 5783

dx.doi.org/10.1021/la300222v | Langmuir 2012, 28, 5783−5794

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small organic compounds with LAA has already been shown to readily increase biological uptake of drugs.14,15 LAAs are α-amino acids containing an alkyl side chain branched to the amine function. LAAs combine the physicochemical properties of both lipids and amino acids due to their amphipathic structure. To our knowledge, there is no data on the use of LAA to improve the intranasal absorption of high molecular weight (>3 kDa) therapeutic proteins. We therefore investigated the ability of using LAA to complex EPO, hGH, and sCT to form colloids that may be sprayed easily. The LAA used in this study is lauroyl proline (LP) and is built by a covalent association between a lauric acid (C12) and a proline as shown in Figure 1D. Our study aims at finding the adequate formulation that preserves the protein structure (controlled by circular dichroism, CD, and molecular dynamics) in a colloidal complex (particle size measured by dynamic light scattering). The structure of the colloidal complex is also studied by molecular dynamics. The passage through nasal cells cultivated at confluence was also investigated.

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MATERIALS AND METHODS

Materials. Recombinant human growth hormone (rhGH) powder (purity 99.7%) containing 87% mannitol glycine, and phosphate buffer were purchased from Auspure Biotechnology (Shangai, China). Amorphous mannitol and glycine guarantee the presence of noncrystallized water linked to the protein, in the freezing step, thus preventing protein denaturation.16,17 Recombinant human erythropoietin (rhEPO) solution at 1.24 mg/mL (purity 100%) containing citrate buffer and sodium chloride was a generous gift from Dragon Pharmaceutical (Vancouver, Canada). Salmon calcitonin (sCT) powder (purity 98.4%) containing 8.2% acetic acid was kindly provided by Welding (Hamburg, Germany). It is also known that calcitonin is less prone to aggregation at acidic pH18 in acetic acid solutions.19,20 Lauroyl proline (LP) was obtained from Seppic (Paris, France). S-MEM and chloramine-T were purchased from SIGMA (Saint Quentin Fallavier, France); 125iodine from Amersham (Buckinghamshire, UK); HBSS from Invitrogen (Cergy Pontoise, France); BEGM KIT from Clontech (Saint-Germain-en-Laye, France); and DMEM medium from Cambrex (Emerainville, France). 24 mm Transwell plates with 0.4 μm pore polycarbonate membrane insert were purchased from Fisher (Illkirch, France); the lactate dehydrogenase (LDH) kit was purchased from Roche (Neuilly surSeine, France), and the epithelial volt-ohm meter (EVOM) was purchased from World Precision Instrument. Preparation of Colloid Suspensions. rhGH, rhEPO, and sCT were dissolved in demineralized water to a concentration of 10 mg/mL (0.45 mM), 1.24 mg/mL (0.036 mM), and 2 mg/mL (0.58 mM), respectively. The protein concentrations were chosen according to preclinical tests planned on sheep. For rhGH, Cheng and co-workers recommend 5 mg/animal.21 Since it is envisaged to use a nasal device delivering 50 μL per spray, this leads to a rhGH concentration of 10 mg/mL. For rhEPO, 250 μg is required, and because the nasal device delivers 200 μL per spray, this leads to a concentration of 1.24 mg/mL. Dua and co-workers recommend administrating 400 μg of sCT per sheep22 so its concentration must be 2 mg/mL. Translucent solutions are obtained for all proteins. Appropriate quantities of dry lauryl proline powder were added to the above solutions to obtain protein/ LP molar ratios ranging from 1/265 to 1/2. At this step, a pH of ca. 3 and an inhomogeneous solution were obtained. The pH was then adjusted by dropwise addition of NaOH 1 N to reach a value of 7−7.4 for rhGH/LP and rhEPO/LP complexes and a pH of 4−5 for the sCT/LP mixture. These pH values appeared the most suitable for protein stability23−25 and also in relation with experiments to be conducted in live animals (vide supra). Protein−LP blends were then stirred during 10 min in order to obtain translucent solutions. Circular Dichroism Measurements. Circular dichroism (CD) spectra were run on a Mark VI Jobin Yvon dichrograph at 0.5 nm

Figure 1. Structure of human growth hormone (hGH) (A), salmon calcitonin (sCT) (B), the human erythropoietin (rhEPO) (C). Images are taken from the Protein Data Bank (numbers 1HGU, 2GLH, and 1BUY). LAA lauroyl proline (LP) (D) was constructed using Chembiodraw ultra 11.0 and Chembio3D ultra 11.0.

been intranasally administered to growth-hormone-deficient patients9 and have produced an increase in bioavailability from 4 to 9%. In rats, the suitability of gelatin microspheres for nasal and intramuscular delivery of sCT was also examined.10 A great hypocalcemic effect was obtained after intranasal administration of sCT-gelatin microspheres. The above works suggest that there may be opportunities to deliver appreciable amounts of proteins into the systemic circulation via the intranasal route. Unfortunately, in most cases, the enhancer caused damage to nasal cells. For example, STDHF promotes in vitro damaging effects on the nasal epithelium, and stops in an irreversible way ciliary movement within a few minutes.11,12 To circumvent these toxicity effects on nasal cells, we use in this study lipoamino acids (LAA) to form colloids that may be easily sprayed and may be prone to improve intranasal absorption of proteins. The toxicity of LAAs has already been tested on biological membranes and biomembrane models. It appears that the toxicity of lipoamino acids depend on the weight of the lipophilic part. LAAs shorter than hexadecanoate showed the best compatibility with various cells, and it was demonstrated that lipoamino acids acted at the cell membrane level. No damaging effects on nucleus or apoptotic induction were observed.13 It is worthwhile mentioning that conjugation of 5784


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intervals over the 180−270 nm range and using 0.1 mm path length cells; four scans were accumulated. Measured CD traces, CDmeas, in degrees, were converted into mean residue ellipticity [θ]mrw, in deg·cm2·dmol−1, using26

[θ]mrw =

3300Δε n

differentiation. The BEGM/DMEM medium mixture was refreshed every day until cells reached confluence. In such a case, a tight junction is formed and a large increase of the system resistivity is observed,32 which can be quantified by measuring the transepithelial electrical resistance (TEER) (vide infra). Transepithelial Electrical Resistance (TEER) Measurement. The integrity of the cell monolayer was determined by measuring TEER values using an epithelial volt-ohm meter (EVOM, World Precision Instrument, Stevenage Herts, England) equipped with chopstick electrodes (Endohm, Brussels, Belgium). Initial conditions are usually defined by considering that cells are at confluence in the monolayer when TEER values greater than 300 Ω·cm2 are measured.33 To be on the safe side, transport experiments were performed when the TEER initial values were close to 1 k Ω·cm2. Analysis of Cell Damage. The integrity of the monolayer was controlled at the end of the experiment by measuring the activity of released lactate dehydrogenase in the extracellular medium (cell-free culture supernatant) with a cytotoxicity detection kit (LDH-Kit, Roche diagnostic, Meylan, France). Passage through Nasal Cells Grown at Confluence. Differentiated cells were washed twice with HBSS medium (Hank’s balanced salt solution, Invitrogen) and were preincubated with the same medium or with demineralized water during 15 min at 37 °C. For experiments with the human growth hormone, 0.5 mL of HBSS containing 0.06 or 0.03 mg/cm2 rhGH (control) and rhGH/LP at molar ratios 1/35 and 1/70 was added to the apical side of cells. After 2 h, the basal side solution was collected and kept at 4 °C until analysis and was quickly replaced by HBSS medium regulated to 37 °C. For erythropoietin, 3% (w/w) NaCl or 15% (w/w) mannitol was added to 0.5 mL of demineralized water in order to increase the osmolarity of the solution. Then 0.06 mg/cm2 rhEPO (control) with rhEPO/LP molar ratios of 1/35 and 1/70 was added to the apical side of cells. After 2 h, the basal side solution was collected and kept at 4 °C for further analysis and then quickly replaced by demineralized water regulated to 37 °C. For calcitonin permeability, 0.5 mL of demineralized water containing 0.06 mg/cm2 sCT (control) with sCT/LP molar ratio of 1/5 was added to the apical side of cells. After 2 h, the basal side was collected and kept at 4 °C for further analysis and then quickly replaced by demineralized water regulated to 37 °C. For all experiments, cells were placed at 37 °C during 15 min, and TEER values were measured to check the monolayer integrity (TEER ≈ 1 kΩ·cm2). The apical-to-basolateral permeability [apparent permeability coefficient, P (cm·s−1)] of each drug was calculated according to the following equation:34

(1)

where n is the number of peptidic bonds (number of residues − 1), and Δε = ε∥−ε⊥, with ε being the molar extinction coefficient. Δε is inversely proportional to the concentration and optical cell path. Dichroic signals of buffer were recorded and shown to give little background, but they were nonetheless subtracted. Samples were allowed to equilibrate for 30 min at a given temperature, regulated to ±2 °C, before the CD signal was acquired. Spectra were smoothed with a 5-point FFT filter before deconvolution with the CDfriend program, developed by S. Buchoux in the laboratory. This algorithm uses standard CD curves of α-helix, β-sheet, helix-II, and random coil obtained from LiKj peptides of known length, secondary structure,27,28 and CD spectrum. CD Pro software (http://lamar.colstate.edu/ ∼ssreeram/CDPro) developed by Woody and Sreerama29 was also used and gave similar results, but CDfriend was preferred because it has less initial guesses. Dynamic Light Scattering. Dynamic light scattering (DLS) is a noninvasive technique used for characterizing macromolecules in solution and particles in suspension.30 The technique measures the time-dependent fluctuations in the intensity of scattered light that occur because particles are undergoing Brownian motions. The velocity of Brownian motions is measured and is called the translational diffusion coefficient (Dt). This diffusion coefficient can be converted into the particle hydrodynamic radius (Rh) using the Stokes−Einstein equation:30

Dt =

kBT 6πηR h

(2)

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity. For eq 2 to be valid, the system has to be diluted so the rhGH-LP and sCT-LP concentrations were lowered to 0.5 mg/mL (respectively 22.5 μM and 145 μM), and rhEPO-LP to 0.12 mg/mL (3.5 μM). The translational diffusion coefficient of colloidal particles was determined by measuring the autocorrelation function at a 90° scattering angle on a ALV/CGS-3 compact goniometer (ALV-GmbH, Germany), and henceforth the mean hydrodynamic radius was computed. The temperature was maintained at 23 °C using a water circulating cryostat during all experiments. As the samples are much diluted, the viscosity of the solution was approximated to that of water. Cell Culture. Nasal culture cells were prepared using a slightly modified Kissel’s method.31 Briefly, nasal cells were obtained during surgery from the inferior turbinate of a patient suffering from rhinitis allergic or septal deviation. The tissues were quickly treated after ablation with protease−DNase mixture (1% protease XIV with DNase) in S-MEM medium (15 mmol/L HEPES, 0.5 mL gentamicine, 2.5 mL penstrep). Incubation was performed for 4 h at 4 °C under gentle shaking. Tissues were then carefully scrapped, and dissociated epithelial cells were washed three times with S-MEM medium. Cells were centrifuged 5 min at 1000 rpm and then diluted in bronchial growth epithelium medium (BGEM) complemented with insulin (2.5 mg/L, 0.4 μM), transferrin (5 mg/L, 0.06 μM), hydrocortisone (0.036 mg/L, 0.1 μM), triiodothyronin (3.5 μg/l, 0.005 μM), epinephrine (0.25 mg/L, 137 μM), hEGF (12.5 μg/l, 94 pM), retinoic acid (50 nM), bovine pituitary extract (15 mg/L), gentamycin BSA-FAF (250 mg/L, 3.7 μM) with antibiotics, and 1% nonessential amino acids. Cells were laid onto coated permeable cell filters in the same medium with a density of 25 000 cells/cm2. After one night in the incubator (37 °C, 5% CO2, 95% relative humidity), cells were washed twice in order to eliminate endothelial cells and resuspended in BEGM medium. The medium was changed every 2 days. After 5−7 days, the BEGM medium was replaced by a mixture of BEGM/DMEM (Dulbecco’s modified Eagle's medium) 1:1 medium supplemented with 0.31 μg/L hEGF in the basal side to induce cell

P=

dC 1 dt AC0

(3)

where dC/dt is the rate of appearance of drugs on the basolateral side (μmol·s−1), C0 is the initial drug concentration in the apical side (mM), and A is the surface area of the monolayer (cm2). Because the presence of lauroyl proline was found to perturb ELISA tests for rhGH and sCT, concentrations of proteins in the basolateral side were determined differently. rhGH was radiolabeled by oxidoreduction reaction with chloramine T, and iodinated rhGH was purified as described by35 on G-100 sephadex chromatography. rhGH concentration was determined by using a liquid scintillation counter (LSC 3500, Aloka, Tokyo, Japan). sCT concentration was determined by HPLC.36 rhEPO concentration was determined by an ELISA EPO kit from Roche (Neuilly sur-Seine, France). Molecular Dynamics. We report here on a set of molecular dynamics simulations that mimic the interactions between two of the experimentally tested proteins (rhGH and sCT) and LP. Because rhEPO had a colloidal behavior similar to that of rhGH, we did not perform simulations to optimize computing time. All the simulations have been done with the GROMACS program,37 at the atomic level, with the GROMOS43.a2 force field,38 using NPT sampling (pressure and temperature are maintained close to standard conditions: T = 300 K, P = 1 bar), with a 2 fs time step and 10 ps (respectively 1 ps) for the pressure (respectively temperature) relaxation time. Topology of LP 5785


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was obtained using the PRODRG program.39 Topology of proteins is obtained from the Protein Data Bank (PDB). For rhGH, two simulation boxes were constructed: the first one with one protein, 32 LP, 15 485 water molecules, 4 sodium ions, and dimensions of 80 Å × 80 Å × 80 Å; the second with one protein, 86 LP, 21 616 water molecules, 4 sodium ions, and dimensions of 90 Å × 90 Å × 90 Å. In the calcitonin case, 8 sCT, 88 LP and 41 944 water molecules, 2 chlorine anions, and box dimensions of 66 Å × 66 Å × 66 Å have been used.

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at 222 and 209 nm and a pronounced positive absorption band at 190 nm that is typical for proteins with a high degree of αhelix content. The PDB indeed reported a high helical content of rhGH crystal (PDB entry: 1HGU).40 Spectral deconvolution of the spectrum in solution gave 67 ± 5%, which is in accordance with the calculated Hα content obtained from the structure found in the PDB and using an internal algorithm (Table 1). Incubation of rhGH under the same conditions but with increasing concentrations of LP showed little modifications for molar ratios 1/35 to 1/70. A more important modification in the band shape was observed on increasing the amount of LP to ratios 1/133 to 1/265. Deconvolution showed that the amount of α-helix decreased and β-sheet increased as Ri varied from 1/ 35 to 1/265 (Table 1). The table shows also that the protein retains most of its structure for Ri = 1/35 and 1/70. For higher amounts of LP, the protein structure was no longer maintained. Figure 2B shows selected far-UV CD spectra for rhGH at pH 7.4 and at similar protein/LP molar ratios. As observed for rhGH, native rhEPO in solution is characterized by a high degree of α-helix content. A high amount of helical content is indeed reported in the crystal (63%). Spectral deconvolution of bands in solution also gives 63% (Table 1). Association of rhEPO with increasing concentrations of LP showed an absorption decrease in the CD signal at 190 nm for molar ratios 1/70 and 1/133. This suggests a loss in α-helix content. Deconvolution showed that the percentage of α-helix indeed decreased as Ri goes from 1/35 to 1/133 (Table 1). As for rhGH, rhEPO retains most of its structure for Ri = 1/35. Figure 2C illustrates the CD spectra of native sCT peptides and sCT/ LP for molar ratios 1/2 and 1/5, in water at pH 5. A ratio sCT/ LP lower than 1/5 has been investigated, but a precipitate was observed. The native sCT and the sCT-LP (1/2) complex showed essentially a negative U type CD spectrum with a minimum at approximately 200 nm, which is generally recognized as representing a state lacking defined structure. Spectral deconvolution gave indeed a high percentage of random coil structure (89%) (Table 1). The sCT-LP (1/5) spectra showed minima at 222 and 209 nm that are indicative of α-helix. Deconvolution showed that the amount of α-helix increases and that of random coil decreased as Ri varied from 1/2 to 1/5 (Table 1). The α-helix content is 40%, a figure very close, within the experimental error, to that reported in the PDB (48%). It is noteworthy that the PDB structure was obtained with NMR in the presence of sodium dodecyl sulfate (SDS). It is clear that LP has a structuring effect on calcitonin. As a summary, the protein/LP ratios that preserve protein structure while keeping a colloidal suspension are 1/35 (1/70) for rhGH and rhEPO and 1/5 for sCT. These preparations where subjected to size determination with light scattering as reported below. Sizes of Protein−LAA Colloidal Complexes from DLS. Figure 3 and Table 2 report the various colloidal sizes obtained with the three proteins in the presence and absence of lauroyl proline. The lipoamino acid prepared alone in solution was also investigated and presents an inhomogeneous size distribution. A cloudy/diffusive suspension is observed by eye inspection and hydrodynamic radii, as determined by DLS, indeed range between 15 and 2000 nm. The formation of some aggregates of LAA is certainly responsible for this inhomogeneous distribution; indeed, it was shown that LAA was able to form aggregates with concentrations lower than the critical micelle

RESULTS

Colloidal Compositions That Preserve Protein Structure. Several trials with different protein−lipoamino acid molar ratios were performed with the aim of obtaining a preparation that is transparent/translucent. This observation ensures that a colloidal suspension is formed whose particle size is small; the size is further checked by DLS (vide infra). Preservation of protein structure was controlled by circular dichroism. Figure 2A shows selected far-UV CD spectra of rhEPO at pH 7.4 and at molar ratios protein/LAA of 1/0, 1/35, 1/70, 1/133, and 1/265. Native rhGH in solution is characterized by minima

Figure 2. Far-UV spectra of rhEPO (1.24 mg/mL) (A), rhGH (10 mg/mL) (B), and sCT (2 mg/mL) (C) with varying protein/lauroyl proline molar ratios. Four scans were accumulated at 23 °C. (A) rhEPO/LP molar ratios were 1/0 (- - -), 1/35 (○), 1/70 (), and 1/ 133 (· · ·), pH 7.5. (B) rhGH/LP molar ratios were 1/0 (), 1/35 (- - -), 1/70 (· · ·), 1/133 (○), and 1/265 (●), pH 7.5. (C) sCT/LP molar ratios was 1/0 (), 1/2 (○), and 1/5 (★), pH 4.5. 5786


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Table 1. Secondary Structure Elements in Percent for Native rhGH, rhEPO, and sCT, in the Crystal, in Solution and in colloidal Suspension with Various Amounts of LP, at 23°Ca protein/LP molar ratio native protein in solution

protein from PDBc

58 18 24

67 16 17

65 11 29

49 29 22

63 13 24

63 14 23

11 0 89

48b 25b 22b

secondary structure

1/265

1/133

1/70

1/35

rhGH

α-helix β-sheet random

34 42 24

33 65 2

55 18 27

rEPO


34 30 36

42 27 31

sCT


1/5

40 4 56

1/2

19 0 81

a Deconvolution of CD spectra was accomplished using the CDfriend Software (S. Buchoux), accuracy ±5%. bSecondary structure of sCT in the presence of SDS. cThe estimate of secondary structure elements from the structures deposited in the PDB is made using the PDB algorithm “display external sequence” (DSSP), accuracy ±10%. Boldface: α-helix content.

concentration (CMC).41,42 Hydrodynamic radii reported in Table 2 are calculated using a distribution in volume that exacerbates large particles. When using a molecular distribution, we obtain a population with a mean size of 14 nm (99.6% of the objects have a size between 13 and 17 nm and 0.4% a size beyond 40 nm). In the absence of LP, rhGH showed a hydrodynamic radius of 2.4 ± 0.2 nm (Table 2). Within the experimental errors, no changes in the hydrodynamic size were observed for a molar ratio rhGH/LP of 1/35 (Table 2). At a molar ratio of 1/70, the data might indicate a slight expansion of the molecule to 2.9 nm (Table 2). For rhEPO, a hydrodynamic radius of 3.8 ± 0.1 nm was observed in solution (Table 2). In the presence of LP, no significant size change was observed (Table 2). sCT in solution had a very small hydrodynamic radius of 0.6 nm (Table 2). An important increase in size was observed in the presence of LP to reach 2.7 ± 0.4 nm at a molar ratio of 1/5 (Table 2). As a general observation, the protein−lipoamino acid complexes were very homogeneous and quite monodisperse in size, with an average radius of 2−4 nm. Calculation of hydrodynamic radii using a molecular distribution gave essentially the same results. Spraying of Colloidal Suspensions. Two systems were tested for spray trials. Spraying trials of the rhGH/LP colloidal suspension were accomplished using a Markos-Méfar nebulizer (Markos-Méfar, Bovezzo, Italy) that is capable to administer from 500 μL to 1 mL in 10 s. The average size of droplets also called mass median aerodynamic diameter is determined at the nebulizer outlet using an API Aerosizer Mach 2 apparatus (Markos-Méfar, Bovezzo, Italy). Sizes ranging from 20 to 30 μm could be successfully obtained using this spray system. For rhEPO and sCT, a BD Accuspray system capable of delivering smaller volumes was tested (BD Medical, Le Pont de Claix, France). Volumes of 150−200 μL could be sprayed, and the size of the droplets estimated to 20−50 μm. Passage of Protein−LP Colloids through a Monolayer Nasal Cell Culture. Two doses have been applied on top of the cell culture, 0.06 and 0.03 mg/cm2. Most significant results have been obtained with 0.06 mg/cm2 (Table 3). In this table, we report only the results for protein/LP ratios for which protein integrity is conserved (vide supra). Comparing “absolute” permeability of proteins led to very different results because the method to test the protein content in the basolateral side of the cell culture is very different. rhGH

has been quantitated by radioactive labeling, rhEPO by immunology, and sCT by HPLC. We will therefore compare relative permeability, with the normalization being made with respect to that of pure protein. The transepithelial electric resistance is not bound to immunology tests but will be nonetheless normalized to the value measured when applying proteins alone, for ease in comparison. Increase in protein passage through cells grown at confluence is therefore assessed by two parameters, increase of P/Pcontrol, where Pcontrol is the permeability measured in the presence of the pure protein, and decrease of TEERcontrol/TEER, where TEERcontrol is the transepithelial electric resistance produced by proteins alone. Concerning rhGH, the colloid preparation leads to little effect on permeability, but the electric resistance decreases by a factor 3−6 (Table 3). rhEPO leads both to a very large increase in permeability (factor 8−12) and decrease in electric resistance (factor 4−5). In the case of sCT, both permeability increase and resistivity decrease are observed but the effect is restricted to a factor of ca. 2. As a conclusion, the colloidal protein/LP complexes led to a global enhancement in protein passage through nasal cells. For all the above experiments, there is no cell damage as the release of LDH is very low and within the experimental error. In the case of EPO, it has been reported that hyperosmolarity (addition of manitol and sodium chloride) may help in crossing the nasal epithelium.43 We have therefore tested the effect of adding 15% manitol or 3% NaCl to our colloidal preparations. Manitol has little effect on permeability and TEER, whereas NaCl potentiates by a factor 1.5−2 the passage already promoted by LP (results not shown). Molecular Dynamics. In order to understand the interactions between proteins and LP at molecular and colloidal levels, we report here on a set of molecular dynamics simulations that mimic the interactions between proteins and LP. Because rhEPO is globular as is the growth hormone and behaves as rhGH in terms of colloidal stability and secondary structure content (vide supra), we only performed molecular dynamics on rhGH and on sCT. We performed calculations using one rhGH and 32 or 86 lipoamino acids. For salmon calcitonin, 8 proteins and 88 lipoamino acids have been considered. The systems are very hydrated (between 15 000 and 42 000 water molecules) in order to be as close as possible to the experimental conditions of colloidal preparations. For both proteins, three to four trajectories have been carried out 5787


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Figure 3. Hydrodynamic radii as obtained from dynamic light scattering on sCT (A), rhGH (B), and rhEPO (C) colloidal systems. The protein/lauroyl proline molar ratio is indicated on graphs. In (D) is also shown the light scattering results on lauroyl proline dispersions. Temperature is 25 °C.

Table 2. Particle Size (radius, nm) for rhGH, rEPO, and sCT in Solution and in Colloidal Suspensions with Various Amounts of LP, at 23°Ca protein/LP molar ratio (dynamic light scattering) rhGH rEPO sCT

1/70

1/35

2.9 3.7

2.4 3.7

1/5

2.7

protein/LP molar ratio (molecular dynamics)

1/2

protein in solution

1/86

1/32

2.0 (2.1)

1.9 (2.1)

2.6

2.4 3.8 0.6

1/11

2.3

a

Accuracy for DLS measurements ranges between 5 and 15%. For molecular dynamics accuracy is estimated to 10%. Figures in parentheses stand for “surface” compared to “random” conditions (without parentheses).

positioned around the protein (i.e., equally distributed in the simulation box so that they do not overlap with the protein, minimum distance > 2 Å), whereas in the “surface” initial conditions, LPs were placed at 2−5 Å from the protein surface. In the first case, the dynamics shows two different regimes.

for durations ranging from 13 to 52 ns (rhGH) and to 500 ns (calcitonin). Because lauroyl proline has a propensity for colloidal aggregates, we have considered two types of initial conditions for the rhGH simulations: “random” and “surface”. With the “random” initial conditions, LPs were randomly 5788


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Table 3. Permeability and TEER Induced by rhGH, rhEPO, and sCT in the Presence or Absence of Colloidal Complexes on Human Nasal Cell Monolayersa rhGH (control) rhGH/LP (1/35) rhGH/LP (1/70) rhEPO (control) rhEPO/LP (1/35) rhEPO/LP (1/70) sCT (control) sCT/LP (1/5) a

permeability after 2 h (10−7 cm2·s−1)

relative increase (P/Pcontrol)

± ± ± ± ± ± ± ±

1.0 0.5 1.2 1.0 12.1 7.9 1.0 1.7

5.4 2.6 6.6 180 2185 1240 0.9 1.4

0.5 0.5 0.5 10 565 30 0.1 0.1

TEER (Ω·cm2) after 2 h 1102 335 174 173 45 35 500 300

± ± ± ± ± ± ± ±

112 30, 439 ± 52 10 12 3 3 32 26

relative decrease (TEERcontrol/TEER) 1.0 3.3 6.3 1.0 3.8 4.9 1.0 1.7

Dose is 0.06 mg/cm2. Temperature is 37°C. Boldface: large increase of permeability or TEER.

Figure 4. Molecular dynamics of human growth hormone/lauroyl proline systems. (A) Initial “random” conditions containing 1 rhGH, 86 LP (green and blue), and solvent (not represented here). Molecules are equally distributed in the simulation box so that they do not overlap with the protein (minimum distance > 2 Å). The color coding of the protein is such that hydrophobic residues are colored in dark pink and hydrophilic in light pink. (B) Final system formed after 25 ns. (C) Initial “surface” conditions, LPs are placed at 2−5 Å from the protein surface, same color codes as in (A). (D) Final system formed after 25 ns.

Initially, LPs tend to form micelles that, on average, contain 10−15 LPs. This process is quite fast (few ns), indicating that LP “concentrations” chosen for calculations (ca. 200 mM) are undoubtedly well above the CMC. Although the CMC of LP is not known, one may compare it to that of SDS (8 mM) and that of stearyl proline (1.75 mM).44 Subsequently, these micelles stick to the surface of the protein, as shown in Figure 4. The dynamics has been pursued further for 50 ns, but the LP micelle adsorbed to the protein did not change much in shape. During the entire simulation, the overall and secondary protein structure remained unchanged.

In the second family of simulations (“surface” initial conditions), there is no micelle formation and most of the LPs directly bind to the protein surface. Interestingly, LPs diffuse onto the protein surface and tend to migrate to more hydrophobic patches to end up with a quite overall capping of the protein. Again, no significant changes in the overall and secondary protein structure of the protein have been observed. Sizes of complexes can be directly estimated from a measure of the protein−lipoamino acid micelle diameter, using a routine in the VMD package (Table 2). To compare with the hydrodynamic radius measured in DLS, we averaged diameters measured on simulations along the x, y, and z dimensions. The 5789


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light pink the hydrophilic ones. The hydrophobic residues are observed to be in contact or oriented toward the LP micelle.

particle sizes estimated from molecular dynamics are systematically smaller than those obtained from DLS because the hydration shell cannot be estimated in the calculations. However, comparable sizes are obtained with the light scattering measurements, suggesting that the picture shown in Figure 4 correctly reflects the colloidal state. The situation is somewhat different with sCT, a much smaller peptide that does not display a well-defined structure in aqueous solution. Preliminary simulations, not reported here, show that the αhelix content of sCT in water is less than 10%, in agreement with CD measurements. Because it was experimentally observed that calcitonin increased its α-helix content upon mixing with LP, we performed three simulations in which initial conditions for each simulation were characterized by a different degree of initial α-helicity: 10% (minimized in water), 40% (from PDB), and 100% (forced α-helix), named T-10, T-40, and T-100, respectively. The three trajectories have the same length: 500 ns. In the course of the dynamics, several folding and unfolding events can be observed for the same peptide, with a rough estimate of the time spent in each conformation of 100 ns. In Figure 5 is shown the average α-helix content for

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DISCUSSION The main result of this study is the finding of appropriate lipoamino acid−protein preparations that markedly increased the permeability through nasal cells. These preparations ensure an active protein structure and are stable under the form of colloidal structures of 5 nm sizes that can be easily sprayed for ease in delivery. These findings are discussed below with possible biological implications. Stable Protein−LAA Colloidal Complexes of Nanometer Sizes. Lipoamino acids are in fact detergents so one can expect that they may unfold globular proteins that have an active structure in solution like rhGH and rhEPO. Evaluation of structural changes caused by the preparation process of proteins with LAA is made through determination of secondary structure content. The α-helix content gives fundamental information not only on the retention of the original structure, but also on the relative activity associated with it.45,46 High doses of lauroyl proline (Ri = 265, 133) led to a large decrease in α-helix content and promoted partial denaturation of proteins. Molar ratios of 70 or 35 practically leave the native helical content unchanged and can be considered as interesting preparations for globular proteins such as rhGH and rhEPO. The case of the smaller peptide calcitonin is somewhat different because it is unstructured in solution. To accomplish its physiological activity, it must interact with receptors under a αhelix conformation. This is usually obtained through a primary interaction with negatively charged phospholipids in close vicinity with membrane receptors.47,48 The sCT/LP (1/5) ratio leads to the induction of a marked α-helix content of ca. 40% and can also be considered as an appropriate preparation. Acquiring helical content in the presence of LAA is in complete agreement with earlier NMR structural studies where the peptide was prepared with a detergent solution of sodium dodecyl sulfate.49,50 Preparations that conserve or promote a high degree of α-helical content have colloidal sizes of ca. 5 nm, as found from both DLS and molecular dynamics. This suggests that the original size of globular proteins is practically unchanged whereas that of sCT is markedly increased due to the association of several peptide monomers with lipoamino acids to form a mixed micelle (vide infra). Interestingly, the lipoamino acid does not make micelles when dispersed in solution but is observed as much larger aggregates of micrometer size. The presence of both proteins and LP lead to nanometer micelles. Such a small size for colloidal preparations is also in favor of a better passage through cellular membranes. Structure of Colloidal Complexes: From Capped Proteins to Mixed Micelles. The intrinsic molecular arrangement of proteins and LP is revealed by molecular dynamics. Concerning the growth hormone, calculations are for certain too short because the end picture varies somewhat depending on initial conditions. The natural tendency for LP to form aggregates is indeed seen in the trajectories when LAAs are closer to each other than closer to the protein (random conditions). Ultimately, the LP micelle that is formed after a few nanoseconds sticks to the protein and a capping of the surface is initiated. This capping is more effective, in the limited time of simulations, when lipoamino acids are initially surrounding the surface of rhGH (surface conditions). It can

Figure 5. 500 ns time course of CT helicity content in calcitonin/LP systems (8 calcitonins, 88 LP, 41 944 water molecules, 2 chlorine anions). Three initial conditions are considered: T-10, calcitonin unfolded, α-helix content 2 Å). At the end of the simulation, a globular structure was obtained (Figure 6B) with LP molecules forming a ca. 4.5 nm diameter micelle covered by sCT that are capping the micelle surface. In Figure 6C, we have represented the inner core of the micelle, exclusively made of LP. In Figure 6D, the proteins are displayed using the following color code: in dark pink are represented the hydrophobic residues and in 5790


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Figure 6. Molecular dynamics of calcitonin/lauroyl proline systems. (A) Initial conditions containing 8 CT (yellow to blue), 88 LP (green and blue), and solvent (not represented here). The α-helix content in each of the proteins is ca. 40% as obtained from the PDB. LP molecules are equally distributed in the simulation box so that they do not overlap with proteins (minimum distance > 2 Å). (B) Final system formed after 500 ns, same color code as in (A). (C) Final LAA-only micelle formed after 500 ns; only LPs are represented here. (D) Final micelle formed after 500 ns, same as (B,) but protein color coding is such that hydrophobic residues are in dark pink and hydrophilic in light pink.

what is observed with globular proteins, the peptides are capping the LP micelle with their lipophilic part facing the lipoamino acids. In fact, the end picture is that of a 5 nm object with a hydrophilic calcitonin nature. Very interestingly, in the complex, calcitonin peptides acquire a marked α-helical character, a configuration that has been shown to enhance activity on receptors.47,55 The mixed micelle has therefore the advantage to present the peptide in adequate structure for action. However, because the surface of the mixed micelle is a protein surface, one may expect that it would not bring great stealth properties (vide infra). It is interesting comparing the forces driving association of lipoamino acids with globular proteins (rhGH and rhEPO) or with the small peptide calcitonin. In both cases, we obtain objects of the same size (5 nm) but with very different topologies. In the case of rhGH, aggregation of LPs with the protein appears to be driven initially by electrostatic interactions (the surface of the LP micelle is rather charged); however, as suggested by the “surface” simulations, nonspecific (hydrophobic) interactions are probably the main factor that governs the capping of the protein by the LPs. Interestingly, the same driving forces have a different effect on the case of sCT. The amphipathic character of both LP and calcitonin (under α-helix fold) appears to drive first the formation of the LP/sCT complex. Electrostatics acts as an additional stabilizing interaction here. It would have been interesting varying the pH of the solutions to explore the role of electrostatics on the association. This is an entire study that was outside the scope of our study. We tried to stay as close as possible to administration conditions on live animals.

be expected that if we could run the simulations for very long times, that is, seconds, both initial conditions would converge toward an entire capping of the globular protein. Although calculations are limited to computer running time, the main picture emerges: the globular protein is capped/covered with lipoamino acids. Very interestingly, the structure of the protein is quasi unchanged in such conditions as also experimentally demonstrated by circular dichroism. The capping phenomenon leads to important remarks: first, the nature of the protein surface has been changed; it resembles to that of a lipoamonoacid surface. This may influence the passage through membranes (vide infra) and increase its stealth properties. It has indeed been shown that the furtiveness of drug-delivery vesicles was obtained by modification of their surface using polyethylene glycol (PEG) chains.51−53 Second, because the size of the protein/LP is very small (5 nm), its stealth properties are also increased. This in accordance with earlier works on nucleic acid delivery, which demonstrated that smaller complex sizes were more efficient in crossing cell membranes.54 Concerning globular proteins, we have only calculated the structure of rhGH with LP. It is reasonable to extend the above conclusions to rhEPO because we have found experimental conditions where the secondary protein structure is quasi unchanged (Ri = 35) and the size of the rhEPO/LP complex is that of the protein alone, within the experimental error. A surface capping of erythropoietin with LP may thus be expected. The case of the smaller peptide calcitonin leads to a completely different picture. A mixed sCT/LP micelle is obtained whose size has increased from ca. 1 nm in the absence of LP to ca. 5 nm in its presence (Ri = 1/5). But at converse to 5791


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Passage through Nasal Cells Increased with LAA. Although there is an intrinsic inaccuracy in permeability measurements due to different methods used to measure concentrations in the basolateral side, permeability increase is nonetheless reported for rhEPO and sCT. TEER measurements that are more robust show a general decrease in cell resistivity for all preparations. The most important effect was observed with EPO where the permeability increased by a factor 8−12 and the resistivity decreased by 4−5 times. For the growth hormone, there was a marked decrease in cell resistivity (3−6 times) but almost not effect on permeability. In the calcitonin case, both permeability and resistivity changed but only by a factor 2. In addition and in the case of globular proteins (GH & EPO), we did not notice differences between the 1/35 and 1/70 ratios that are appropriate in keeping the protein secondary structure unchanged. Although cell passage was largely increased by the presence of LP, it is however difficult to conjecture where is occurring the passage. There may be at least two possibilities: (i) passage through tight junctions or (ii) passage across lipid membranes. The second mechanism is related to some kind of light detergent action or facilitated endocytosis/exocytosis and could be facilitated by the presence of LP that has some detergent character. Interestingly, this occurs without killing cells because they have been checked to remain intact in our conditions. Passage through tight junctions may be favored by hyperosmotic solutions of NaCl, as observed for rhEPO. Such a NaCl effect has been already reported56 and described as losing the junctions and favoring passage through intercellular spaces. One can even envisage a synergism of the two mechanisms because conjunction of both LP colloids and NaCl leads to very important protein passage for rhEPO (increase of permeability by a factor of ca. 20 and decrease of TEER by a factor of ca. 10). Another interesting side result is the fact that ELISA tests could not work well to determine protein concentration in the basolateral compartment for rhGH and sCT. This indicates that proteins remain under a LP colloidal complex, after cell passage, because there are not recognized by immunology. Such a situation is not observed for EPO, suggesting that at least the binding site is not capped by lipoamino acids and allow immunology to work. As a summary, passage through cells is largely favored by LP but the minute mechanisms require further investigations, which was clearly out of the scope of the present study. Biological Implications. Drug delivery is a very complex field, and several approaches have been pursued in trying to deliver molecules, most of them being based on large cargo entities (liposomes, polymer capsules, etc.) being able to trap in their interior active molecules. Although our study falls into this very large field, it is however restricted to the fact that the proteins used herein already work when applied as intravenous injections. Our approach aimed at forming small proteic entities that have different surface properties and that may be sprayed for nasal delivery. The very small size of the colloidal preparation and the new surface properties may work together in helping cross membranes or cell junctions and increasing the furtiveness of the nanoobject that would otherwise be prematurely destroyed by the immune system. Our approach that was restricted to the growth hormone, erythropoietin, and calcitonin could be extended to other proteins and other pharmaceutical drugs. However, our systems remain to be validated at the in vivo scale by applying the protein colloidal complexes to animals and humans.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: CBMN, UMR5248 CNRS, Université Bordeaux, IPB, Allée Geoffroy Saint Hilaire, 33600 Pessac, France. Telephone: +33 5 4000 6818. E-mail: e.dufourc@iecb. u-bordeaux.fr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are very grateful to ANRT and CIFRE systems for funding. CNRS, University of Bordeaux, and the Aquitaine Government are thanked for equipment support.

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REFERENCES

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