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Colloids and Surfaces B: Biointerfaces 133 (2015) 140–147

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb

iatom-inspired skeletonisation of insulin – Mechanistic insights intorystallisation and extracellular bioactivity

iosángeles Soto Véliza, Catharina Alamb, Thiago Nietzela, Rebecca Wyborskia,dolfo Rivero-Müllerc,d, Parvez Alama,∗

Laboratory of Paper Coating and Converting, Centre for Functional Materials, Abo Akademi University, Porthaninkatu 3, 20500 Turku, FinlandBiomedical Science Research, Turku, FinlandDepartment of Physiology, Institute of Biomedicine, University of Turku, Turku, FinlandDepartment of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093 Lublin, Poland

r t i c l e i n f o

rticle history:eceived 26 March 2015eceived in revised form 24 May 2015ccepted 31 May 2015vailable online 9 June 2015

a b s t r a c t

In this paper, we encage insulin within calcium carbonate by means of a biomineralisation process.We find that both dogbone and crossbone morphologies develop during the crystallisation process. Thecrystals break down into small nanocrystals after prolonged immersion in phosphate buffer solution,which adhere extracellularly to mammalian cells without causing any observable damage or early cell-death. The mechanisms behind calcium carbonate encaging of single insulin monomers are detailed. This

eywords:alcium carbonaterystallisationirect encapsulation

nsuliniatom

communication elucidates a novel, diatom-inspired approach to the mineral skeletonisation of insulin.© 2015 Elsevier B.V. All rights reserved.

iabetes

. Introduction

Insulin dependent diabetes mellitus, commonly known asiabetes type 1, has been described as the immune-mediatedestruction of endocrine �-cells located in the pancreatic isletsf Langerhans [1]. The selective destruction of �-cells results innsulin deficiency in the body and induces specific microvascularathologies such as neuropathies, nephropathies and cardiovascu-

ar diseases increasing the risk of mortality [2–8]. Therefore, insulinreatments are needed in order to control blood glucose levels andrevent further complications due to hyperglycaemia [9].

To date, the most common treatment has been daily subcu-aneous injections of insulin which disrupts a normal life. As aesult, in the past decade, pharmaceutical companies have been

ttempting to develop non-invasive delivery systems capable ofimicking the insulin secretion of �-cells [10]. Breakthroughs

n short-term diabetic treatment include technologies such as

∗ Corresponding author at: Adjunct Professor of Composite Materials andiostructures, Laboratory of Paper Coating and Converting, Centre for Func-ional Materials, Abo Akademi University, Porthaninkatu 3, 20500 Turku, Finland.el.: +358 22154858.

E-mail addresses: parvez.alam@abo.fi, shantanou@gmail.com (P. Alam).

ttp://dx.doi.org/10.1016/j.colsurfb.2015.05.047927-7765/© 2015 Elsevier B.V. All rights reserved.

Exubera®, a commercialised pulmonary delivery system, laterremoved from the market [11], and EligenTM (Emisphere technolo-gies), an ongoing project for oral insulin delivery based on the useof synthetic non-acylated amino acids as carriers [12].

1.1. Biomineralisation

Drug delivery systems should ideally be designed as functionalcarriers with specific sizes, morphologies and with desired chemi-cal characteristics, whilst retaining the functionality and bioactivityof the drug. One way this can be achieved may be through deriv-ing inspiration from natural processes of biomineralisation [14].Biomineralisation gives birth to a broad range of minerals withfunctional patterns and diverse properties. It does so by tailoringthe formation of complex inorganic–organic structures, where theorganic component acts as a template to control mineral nucleationand crystal growth [15,16].

Diatoms are inspirational models for biomineralisation. Theseare a group of eukaryotic algae characterised by their unmatched

and so far, irreproducible hierarchical frustules (silica exoskele-tons). Their beautiful shells are a result of genetically guidedbiomineralisation where the cell is encaged within a protectiveskeleton [17]. These skeletons are amorphous, species-specific

ces B:

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D.S. Véliz et al. / Colloids and Surfa

iomineral structures with diverse features including: large surfacerea, ornamented porosity and hierarchically ordered three-imensionality [18]. These characteristics potentialise diatomrustules in biocarrier applications for drug and gene delivery, ands silica-based biohybrids [19–22].

.2. Calcium carbonate as a delivery system

Calcium carbonate has been long considered an effective matrixor delivery systems [23–26], having been loaded as microcapsules,ith biomacromolecules such as insulin and bovine serum albu-in (BSA) [27]. Direct encapsulation of biomacromolecules into

alcium carbonate particles by either interfacial reaction using n-exane solution [44] or through simple mixing [45] is also proveno be highly efficient and comparable to similar methods usingilica microcapsules [44]. Biomacromolecules used in direct encap-ulation include primarily BSA, duplex DNA and betamethasonehosphate.

Bioinspired methods have long been used in conjunction withalcium carbonate to control morphogenesis [28–33]. Furthermore,he polymorphology of calcium carbonate can be guided through itsombination with different additives. Some of the more commonlytudied additives include magnesium, PVP-SDS complex micellesnd different amino acids [34,35,28,36–42,15]. Composite crystalsesulting from the interactions between additives and mineralsevelop different shapes such as rod-shaped, cube-like, hexagon-

ike, cauliflower-like and spinose spheres [16,43]. In each case thehape is guided by the biopolymer templates, which themselvesecome skeletonised by the mineral. Yet, the interactions betweenrganic templates (or growth modifiers) and the crystals are stillot well understood.

.3. Objectives

To date, insulin has been loaded into manufactured carriers46,47] by adsorption or co-precipitation methods [48–50]. Con-rary to this approach, we intend to ‘grow’ a mineral skeleton-likeage around and between insulin molecules, similar in concept toow a diatom frustule encages an underlying cell. In this sense,alcium carbonate crystallisation will be guided by an insuliniopolymer template to form an insulin delivery particle. The pro-osed method potentialises (a) the energetic stability of insulinithin the delivery particle, (b) the preservation of insulin func-

ionality, and (c) the long-term entrapment of insulin with aontrollable rate of release. Herein, we endeavour to elucidate fun-amental mechanisms involved in the skeletonisation of insulin byaCO3 and in its subsequent attachment to mammalian cells.

. Methods

.1. Crystallisation and characterisation of CaCO3/insulinarticles

Calcium carbonate particles were precipitated in the presencef insulin molecules. The precipitation reaction was carried out byissolving 0.175 g of calcium nitrate tetrahydrate (Sigma–Aldrich)

n 150 ml of deionised water. In a second beaker, 0.175 g of sodiumicarbonate (ReagentPlus®, Sigma–Aldrich) was dissolved in 10 mlf deionised water. Insulin lispro (HUMALOG 100 U/ml, Eli Lilly)as immunolabelled with goat anti-insulin (Santa Cruz Biotech-ology) followed by donkey anti-goat antibody conjugated to Alexa

luor® 488 (Molecular Probes), after which it was also added to theecond beaker. HUMALOG® insulin lispro also contains glycerine,ibasic sodium phosphate, metacresol, zinc ions and traces of phe-ol and water. Both solutions were mixed together and agitated

Biointerfaces 133 (2015) 140–147 141

using a magnetic stirrer for 30 min. Under agitation, NaHCO3 reactswith Ca(NO3)2 · 4H2O to form a precipitate of CaCO3.

The crystals were immersed into a phosphate buffer solution(PBS). Prior to any tests involving the crystals, they were washedthree times with deionised water after which they were air driedat room temperature overnight (23 ◦C and 55% RH (relative humid-ity)). Samples of calcium carbonate particles were taken from thePBS solution weekly and imaged using a scanning electron micro-scope (SEM, Jeol JSM-6335-F). To characterise the original crystalmorphologies, crystal imaging was also performed on the first dayprior to PBS immersion and washing. Energy-dispersive X-ray spec-troscopy (EDS) and Fourier transform infrared spectroscopy (FT-IR)were also conducted for the purpose of chemical characterisation.

2.2. Bioactivity of CaCO3 skeletonised insulin: confocalfluorescence microscopy, fluorescence microscopy

To verify the presence of labelled insulin within the particu-lates, we used an antibody against insulin and a secondary antibodylabelled with Alexa Fluor® 488 for visualisation. Alexa Fluor® 488is a green-fluorescent dye that is excited at a wavelength around488 nm (max 490 nm) and results in a maximum emission of525 nm. Microscopy was performed using a Zeiss LSM510 METAconfocal microscope.

Interactions between the particulates and living cells followedprolonged immersion in PBS. We characterised these interactionsby placing the insulin-containing particulates into cultured HEK293(Human Embryonic Kidney Cells) cells. These cells are known tohave insulin receptor sites [51].

The culture plates with both particulates and cells were imagedby fluorescent microscopy within the 1st minute, at 5 min, at 4 hand 1 week after addition to culture plates. EVOS fluorescent micro-scope (Life Technologies) was used for the fluorescent imagingusing the GFP filter.

2.3. Atomistic mechanisms of crystal growth

Molecular dynamics (MD) simulations (Ascalaph Designer)were used to understand the specific interactions between theinsulin molecules and the calcium carbonate particles. The insulinmolecule was built using the HUMALOG® insulin amino acidsequence, which differs slightly from human insulin in that thereis a switch in the amino acids of lysine (from B29 → B28) with pro-line (from B28 → B29). Calcium carbonate molecules were built andquantum mechanically optimised using the Firefly QC package [52],which is partially based on the GAMESS (US) [53] source code. Weoptimise by electrostatics MP2 with 6-311 + G(2d,p), since Gauss-ian basis sets have been previously used during calcium carbonatesimulations [54–56]. The simulations were stopped when the inter-molecular energies reached steady state.

The force field chosen for the simulation was AMBER94 becauseit has been recommended for protein modelling [57] and because itfocuses on the analysis of intermolecular and intramolecular inter-actions [58]. The united-atom force field model of AMBER94 (Eq.(1)) includes [59]: (a) harmonic potentials to model angle terms,(b) Fourier series to represent the torsion terms, (c) 6–12 potentialsfor the van der Waals terms, and (d) Coulombs law to calculate theelectrostatic terms.

Etotal =∑bonds

[Kb · (b − beq)2] +∑

angles

[K� · (� − �eq)2]

+∑

dihedrals

Vn

2· [1 − cos(n� − �)] +

∑i<j

[Aij

(Rij)12

− Bij

(Rij)6

+ qiqj

�(Rij)12

]

(1)

142 D.S. Véliz et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 140–147

Fi

caapaaqlm

3

3m

aca

lffl

ig. 1. Confocal microscopy images of calcium carbonate crystals grown under thenfluence of insulin. Scales: 50 �m (upper) and 10 �m (lower). Images taken = 10.

The terms in the equation for the bond stretching are the forceonstant (Kb), bond length (b) and equilibrium value (beq). For thengle bending, the terms are the force constant (K�), bond angle (�)nd equilibrium value (�eq). The torsion terms considered includeeriodicity (n), force constant (Vn), torsion angle (�) and phasengle (�). Finally, the non-bonded interactions between the i and jtom pairings include Aij and Bij as van der Waals parameters; alsoi and qj for the atomic charges, R for the non-bonded interactionength and � as the dielectric constant including the effect of the

edium.

. Results and discussion

.1. Characterisation of skeletonised insulin by FT-IR and confocalicroscopy

Insulin is a complex protein structure with sites that may attractnd template the precipitating calcium carbonate during the pro-ess of precipitation, in similitude with other proteins, amino acidsnd polymeric additives [35,28,36–42].

To confirm that CaCO3 particulates precipitated withoutabelled insulin do in fact, not fluoresce green, we initially per-ormed confocal microscopy of these insulin-free particulates. Nouorescence was detected in the images. Fig. 1 shows confocal

Fig. 2. FTIR results from top to bottom: calcium carbonate, insulin and calciumcarbonate crystal grown under the influence of insulin. FTIR repeated twice.

images of the insulin–CaCO3 particles. The images confirm thatthe CaCO3 particles are laced with labelled insulin, since in theseimages, the particles can be seen to fluoresce green.

Fig. 2 shows FTIR spectra for pure precipitated calcium car-bonate, insulin and the insulin–CaCO3 crystals. The pure calciumcarbonate shows typical strong peaks at 1433 cm−1 and 874 cm−1

(including small peaks at the base at 856 and 846 cm−1); it alsoshows weaker peaks at 1090 cm−1 and a double peak (also weak)at 746/712 cm−1. Previously published IR data includes character-istic absorption bands for calcite at 1420, 876 and 714 cm−1 andfor metastable vaterite at 1090, 878/850 cm−1 and 747/741 cm−1

[60]. Therefore, the reaction between sodium bicarbonate and cal-cium nitrate yields CaCO3 precipitates of calcite and metastablevaterite. The slight differences between the peak values can beattributed to that our samples are composites of the polymorphsof CaCO3 as opposed to pure samples. Differences in the prepara-tion methods may also alter the IR spectra somewhat [35,61]. Nopeaks were detected for sodium carbonate suggesting that sodiumions do not play any definitive role in the precipitation process.Pure insulin shows characteristic absorption bands for polypep-tides including strong peaks at 1644 cm−1 ( form, C O stretching,amide I band) and 1538 cm−1 ( form, NH deformation vibration,amide II band), and a medium wide peak at 3295 cm−1 (NH stretch-ing intramolecular hydrogen bonded) [62].

The insulin–CaCO3 particulates showed very strong peaks at1411 cm−1 and 871 cm−1, a medium peak at 1058 cm−1 and var-ious weak peaks of different widths at 3304 cm−1, 1660 cm−1 and713 cm−1. Overall, the absorption bands are representative of cal-cium carbonate in the form of calcite [60]. The spectrum obtainedfor the insulin–CaCO3 particulates is a result of the influence of theinsulin molecules over the calcite spectra, as it can be seen in Fig. 2.Arrows in the figure show peaks from both insulin and calcium car-bonate. Moreover, the strong concave peak of calcite (1433 cm−1)is a strong but convex peak (1411 cm−1) once the insulin has beenskeletonised. The broad peak in the spectrum at 1065 cm−1 is iden-tified as phosphate [63,64]. There is no phosphate in the insulinspectrum since pure dried insulin was used for testing; but dur-ing crystal formation, HUMALOG® was used which contains dibasic

sodium phosphate.

On degradation, the absorption bands for the calcium carbon-ate particles change considerably. Fig. 3 represents the absorptioncurves for samples taken over 3 weeks. They are all similar and

D.S. Véliz et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 140–147 143

Fe

s1cauapratcu1itc

ckaaadwic

3

oot

roTad

Fig. 4. SEM images of calcium carbonate crystals in PBS from weeks 0 to 12. An EDSimage showing the crystal elements spectrum with a dominant calcium (green)presence. Scales: 10 �m (weeks 0–2) and 1 �m (week 12). Minimum number of

ig. 3. FTIR results for crystals after week 1, week 2 and week 3. FTIR repeated twiceach week.

how a strong peak around 3330 cm−1 and a medium peak around635 cm−1 which indicates the presence of amorphous (‘hydrated’)arbonate minerals [65]. The conformational change from calcite tomorphous calcium carbonate (ACC) occurs once the nanopartic-lates are immersed in PBS. A reaction between the particulatesnd the ions dissociated within the solution is unlikely since thearticulates were stable prior to PBS immersion. For a substitutioneaction to occur, energy is needed to break the calcium carbonatepart after which, further energy is required to activate substitu-ion, which makes it highly unlikely. In fact, transformation fromalcite crystals to hydroxyapatite has been achieved in laboratoriessing hydrothermal conversion which require temperatures over20 ◦C and high phosphate concentrations [66]. The FTIR spectrum

n our samples confirms there is no phosphate in the crystals afterhe immersion; however, there is an evident influence of PBS sincealcium carbonate particulates become amorphous.

Insulin may act as an organic template for the crystallisation ofalcium carbonate, which suggests that so long as the particles areept in solution and not dried, the insulin will remain skeletonisednd continue to interact strongly with the calcium carbonate. Zhaond Wang [15] reported that on drying, templating macromoleculardditives (in their case PVP-SDS complex micelles) can detach andegrade leaving hollow CaCO3 crystals. Based on the work of [15],e also suggest that once dried, biomacromolecule/particulate

nteractions may start to break down leaving eventually, hollowrystals of calcium carbonate.

.2. Crystal morphologies of skeletonised insulin

Degradation and particulate break-down is an important qualityf drug delivery vehicles since it allows for the controlled releasef drug over time. Fig. 4 shows the morphologies of CaCO3 skele-onised insulin as a function of immersion time in PBS.

For the first week from the point of the initial precipitationeaction, dogbone and crossbone crystal morphologies are clearlybservable. These are ca. 5 �m in length and ca. 1 �m in diameter.

he crystals start to either break up into nanoparticles or degrades a function of immersion time in PBS. Many of them lose theirogbone/crossbone shapes when they break up into nanoparticles,

images taken each period = 5. (For interpretation of the references to colour in text,the reader is referred to the web version of the article.)

however even after three months some crystals under high magni-fication (×4000) can still be seen to retain the dogbone morphology.

Particles with dogbone-like morphologies are often referred toas ‘dumbbell’ in the literature. To date, dumbbell shapes have onlybeen observed to occur in calcium carbonate crystallised with aspecific ratio of magnesium and malic acid [67], and in gel-growncalcites [68,69].

We suggest thus that certain functional groups of the aminoacids making up insulin act as nucleation sites and may guide crys-

tal morphology. Comparing the amino acids of insulin against themalic acid used by Meldrum and Hyde [67], to promote dumbbellgrowth, Fig. 5, we find that the glutamic acid of insulin has a similar

144 D.S. Véliz et al. / Colloids and Surfaces B:

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Fig. 5. Amino acid from insulin compared to malic acid.

tructure to malic acid. Both molecules are substituted dicarboxyliccids, which according to Meldrum and Hyde [67] is responsibleor developing dumbell shapes. The growth and final morphologyf crystals in the presence of additives will be influenced by therystal face that the additives template to. Dicarboxylic acids tendo template to (1 0 4) CaCO3 crystal faces [43] and typically theseaces are terminal faces, in that further growth is retarded. By tem-lating the (1 0 4) faces, precipitation and growth is able to continue

n a direction perpendicular to these faces and as a result, crystalsecome elongated.

Amino acids can direct nucleation and therefore, guide andefine the growth, size and morphology of crystals [71]. The addi-ion of peptides into the crystal system causes changes in therientational ordering of the crystallites once crystallisation ini-iates and kinetic factors (such as increasing surface energy) leado tilting between crystallites; resulting in curved morphologies70,43]. Curved surfaces are hence typical to peptide–CaCO3 com-osite crystals.

We suggest that at specific bonding sites, insulin attracts Ca2+

ons through primarily electrostatic interactions and hydrogenonding. These in turn create nucleation sites for further calci-cation over the insulin molecules. Amino acids of the insulinolecule containing a high number of carboxylic groups (especially

icarboxylic acids such glutamic acid) develop an elongated mor-hology in calcium carbonate crystals [43,67]. Amino acids withNH+

2 and COO− functional groups act as nucleation sites for cal-ium carbonate crystals [38] and amino acids with OH functionalroups take part in controlling morphology during crystallisation56].

.3. Molecular dynamics simulation

Prior to CaCO3 addition the HUMALOG-insulin was allowed toold to an energetic steady state within implicit water conditions.alcium carbonate molecules were added one at a time to the sim-

lated insulin molecule. Almost 200 calcium carbonate moleculesere required for the full coverage of the insulin molecule. There

re noticeable regions on the insulin molecule that have a highernd lower affinity to calcium carbonate.

ig. 6. Pictographic summary of the encaging of insulin molecules. Colours in the insulgreen). Encaging starts with the attraction of calcium ions in high affinity sites and repuls

olecule. Further addition of calcium carbonate favours complete skeletonisation of theolour in text, the reader is referred to the web version of the article.)

Biointerfaces 133 (2015) 140–147

Calcium carbonate molecules were repelled from specificregions of the insulin molecule, subsequently attaching to adifferent region of the molecule. We find that aromatic amino acidssuch as phenylalanine (Phe) and tyrosine (Tyr) are mainly respon-sible for calcium carbonate repulsion. The amino acids valine (Val),leucine (Leu) and isoleucine (Ile) also show a low affinity to calciumcarbonate to a lesser extent. In contrast, glutamic acid exhibited anexpectedly high affinity to calcium carbonate [56]. Further, calciumcarbonate addition showed that CaCO3-‘bridging’ occurs over thelower affinity regions. This is where additional calcium carbonatemolecules connect to CaCO3 over regions of insulin that are repul-sive to CaCO3. The result is that the CaCO3 forms a shell that encagesthe insulin though the shell is both attached to and detached fromthe insulin molecule, Fig. 6.

A review of the primary distances between shell/insulinmolecules and observations during simulation show molecularguiding of the CaCO3 regarding the high and low affinity regions.Glutamic acid has the lowest average O Ca (amino acid–CaCO3)distance overall (2.5 ± 0.2 A). Glutamic acid therefore attractscalcium molecules through its carboxylate functional groups. Inter-action between the aromatic ring in amino acids and calciumcarbonate particles has an average H Ca (aromatic ring–CaCO3)distance of 3.6 ± 0.2 A. Nevertheless, aromatic amino acids haveshort H O (amino acid–CaCO3) distances (2.8–3.1 ± 0.2 A). Tyro-sine is an exception, in that its hydroxyl has an average O H(amino acid–CaCO3) distance as low as 1.6 ± 0.2 A. H Ca distances(aromatic ring–CaCO3) indicate that aromatic amino acids repelcalcium ions due to their non-polarity. Furthermore, short O H(amino acid–CaCO3) distances are the result of the observed reloca-tion of calcium carbonate molecules. Repelled molecules are guidedtowards adjacent calcium carbonate molecules and the carbonateend of the molecule is shifted towards the aromatic ring.

Therefore, in order to develop a complete shell around the lowaffinity regions of insulin (‘bridging’), calcium carbonate moleculesneed to reshuffle such that the calcium ions are not closest to theamino acids, yet are still able to connect to the surrounding CaCO3molecules.

3.4. Biological interactions

Biological interactions are used to understand the bioactivity ofencaged insulin and further cell response to the CaCO3 particles.The nanoparticulates were initially scattered randomly in the cellculture. After 4 h some of the nanoparticulates had migrated andadhered to cell surfaces, as seen in Fig. 7(a) and (b). The number

of nanoparticulates continued to attach to the cells as a functionof time despite concurrent nanoparticle degradation in the immer-sion medium. The green fluorescence also confirms that insulin isstill well integrated within the calcium carbonate. It may also be

in molecule represent very high affinity sites (orange) and very low affinity sitesion from the low affinity amino acids, creating a shell with holes around the insulin

molecule until a full coverage is achieved. (For interpretation of the references to

D.S. Véliz et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 140–147 145

Fig. 7. Fluorescence microscopy images after 4 h (a) and 4 days (b). Scale: 100 �m, and fluorescence microscopy image overlay after 4 days of calcium carbonate particlesa ach p

pfisTAtwsst

4

itlim

•

•

•

ddition to the cell cultures (c). Scale: 100 �m. Minimum number of images taken e

ossible, that free regions of insulin, where the CaCO3 has suf-ciently degraded, attach to appropriate extracellular receptorsites, Fig. 7(c) via the classical binding surfaces (Ile A2, Tyr A19,yr B16 and Tyr B26) and the second receptor binding surfaces (Ser12, Leu A13, Glu A17, His B10, Glu B13 and Leu B17) [72], provided

he solution within which it is in is close to its isoelectric point. Theidespread distribution of insulin about the broken nanocrystals

uggests that in the initial precipitation reactions, insulin is in factkeletonised within the particulates, and does not simply attach tohe surfaces of the precipitated particle.

. Conclusions

Calcium carbonate particles were precipitated in the presence ofnsulin. The crystals were morphologically and chemically charac-erised. Dogbone and crossbone shaped calcite formed with initialengths of ca. 4–5 �m. Interactions between calcium carbonate,nsulin and living cells were also studied. Combining the experi-

ental and modelling data, we are able to conclude the following.

During crystallisation, amino acids such as glutamic acid cre-ate a high affinity between the calcium carbonate molecules andinsulin, and create nucleation sites for further CaCO3 precipita-tion.Aromatic amino acids in the insulin repel calcium carbonate.Nevertheless, interactions between CaCO3 permit the growth of

CaCO3 bridges over these regions. This process results in the com-plete skeletonisation of insulin.Crystals containing insulin attach to cell surfaces with no visibledamage to the cell.

eriod = 3.

• Degradation over time should give rise to a gradual release ofinsulin from the crystals that is potentially bioactive. This will beconfirmed in future experiments.

Acknowledgements

We thank Dr. Diana Toivola and Professor Martti Toivakka forfacilitating some of the equipment used in this research.

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