Journal of Periodontology; Copyright 2013 DOI: 10.1902/jop.2013.120679

1

Peptides:β-Cyclodextrin Inclusion Compound as Higher Effective Antimicrobial and Anti-epithelial Proliferation Agents

Jessika Consuegra*, Maria Elena de Lima

†, Daniel Santos

†, Rubén Dario Sinisterra

‡, Maria

Esperanza Cortés§

*Department of Physiology and Biophysics of Biologic Science Institute - ICB, Federal

University of Minas Gerais, Belo Horizonte – MG, Brazil.

†Immunology and Biochemistry Department of Biologic Science Institute - ICB, Federal

University of Minas Gerais, Belo Horizonte – MG, Brazil.

‡Chemistry Department, Exacts Science Institute, Federal University of Minas Gerais, Belo

Horizonte – MG, Brazil.

Background: The use of antimicrobial peptides (AMPs) as therapeutic agents for periodontal infections has

great advantages, such as its broad spectrum of action, low toxicity, and limited bacterial resistance. However, its

practical use is limited due to the large amount of peptide required to exercise its microbicide function.

Methods: LyeTxI, LL37f, and KR12 cationic peptides were prepared with β-Cyclodextrin (βCD) at 1:1 molar ratio. The susceptibility of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and

Fusobacterium nucleatum were assessed in anaerobic conditions. Cytotoxicity assays were performed using

osteoblast and Caco-2 epithelial cells, and hemolytic activity on rabbit erythrocytes at an absorbance of 414 nm.

Parameters of roughness and electrical charge of membrane were established by Atomic Force Microscopy (AFM)

and Zeta potential, respectively.

Results: The AMPs/βCD decreased drastically the peptide concentration required for activity against

bacteria tested. Moreover, AMPs associated to βCD were able to modify the cell surface parameters, such as

roughness and zeta potential. On the other hand, AMP/βCD did not alter the degree of hemolysis induced by the pure

AMPs. The ECs50 values of the peptides and compounds on osteoblast were greater than the concentrations required

to achieve inhibition of bacterial growth in all the species tested. The AMP/βCD inhibited the proliferation of Caco-2

epithelial cells in a more efficient manner than AMPs solely.

Conclusion: AMPs/βCDs are more efficacious to inhibit periodontopathogenic bacteria with additional

ability of inhibiting the proliferation of epithelial cells at non-cytotoxic concentrations for osteoblasts and

erythrocytes.

KEY WORDS:

Antimicrobial Cationic Peptides, Periodontitis, Cyclodextrins, Cell proliferation, Caco-2 Cells.

Periodontal disease involves complex interactions between microorganisms in the oral cavity and

host factors such as immunological and genetic differences, which cause alteration of connective

tissue homeostasis and clinical manifestations of the disease 1, 2

. The periodontium clinical

condition is associated with the presence of different types of microorganisms, including gram-

positive cocci under healthy conditions, anaerobic gram-negative bacteria in the most pathogenic

state and enteric gram-negative rods as over-infecting bacteria 3-6

. The polymicrobial biofilm and

consequent inflammatory response are the causes of periodontal disease, which makes

elimination of biofilms crucial for treating periodontitis as well as reducing the local and

systemic inflammatory load. Conventional treatment of periodontal disease includes scaling and

root planing (SRP), which serves to mechanically disrupt the biofilm and remove the dental

calculus, thereby reducing the quantity of periodontal pathogens and increasing the number of

microorganisms compatible with oral health 7, 8

. However, mechanical treatment does not

Journal of Periodontology; Copyright 2013 DOI: 10.1902/jop.2013.120679

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eradicate all pathogenic species 8. After SRP of a periodontal pocket wound healing generally

leads to the formation of a long junctional epithelium but not to regeneration of an original

periodontal attachment 9, 10

. As a strategy to prevent the oral re-colonization by periodontal

pathogens after SRP, the mechanical therapy is used in combination with antibiotics; this therapy

has shown the reduction of microbial load, which provides a greater gain in clinical attachment

levels and reduction of the probing depth 11

. However, microorganisms have developed antibiotic

resistance factors which, in an organization of polymicrobial biofilms, cause a reduction in

antibiotic susceptibility 12, 13

.

Antimicrobial peptides (AMPs) that are active against gram-negative and gram-positive

bacteria as well as fungi, have emerged as a new treatment alternative to overcome the

microorganisms resistance to conventional medications 14

. AMPs are amphipathic, positively

charged, short peptides that exhibit antimicrobial activity and play an important role in the innate

immune response. AMPs interact with lipids of the microbial cell membrane, thereby shifting and

modifying the structure of the membrane. The interaction also induces pore formation and, in

some cases, allows for entry of these peptides into the target cell 15

.

The LL37 peptide, part of the family of cathelicidins, is positively charged (+6) at

physiological pH 7.4, and has a hydrophobic N-terminal domain and α-helix conformation. LL37

is secreted to the skin surface and then processed by a serine protease into three subsequent

peptides, each with antimicrobial activity. The smallest peptide is KR-20, which is referred to as

LL37f in this study. The cleavage products of LL37 have lower hemolytic activity, a reduced

ability to stimulate the secretion of IL-8 from keratinocytes, and the highest antimicrobial activity 16

. The smallest fragment of LL37 that has antimicrobial activity has been identified as KR12

which is residues 18-29 of LL37. This peptide has a selective effect against bacteria and is not

cytotoxic to human cells below a concentration of 100 μg/mL 17

.

The LyeTxI peptide isolated from the venom of the spider Lycosa erythrognatha has

antimicrobial activity against Escherichia coli, Staphylococcus aureus, and fungi 18

. The

mechanism of action of LyeTxI seems to be related to the formation of pores in the membrane, as

evidenced by studies with liposomes 18

. Some bacterial species have developed resistance to

AMPs by changing the surface charge, limiting peptide interaction through the membrane

capsule, and producing proteolytic enzymes that may inactivate AMPs 19, 20

.

The use of antimicrobial peptides as therapeutic agents for bacterial infections has a number

of advantages, such as its broad spectrum of action, low toxicity, and limited bacterial resistance,

although some bacteria have developed resistance. However, its practical use is limited due to the

high concentration of peptide required to exercise its microbicide function, which makes the

production of pharmaceutical formulations a very costly process. As a strategy to avoid these

limitations, we have proposed an inclusion compound between the AMPs and β-cyclodextrin

(βCD), because these inclusion systems with peptides on CDs has been shown to improve

solubility and stability as well as decrease protein aggregation and precipitation, which could be

the result of stabilization of the functional form of the native protein in solution 21, 22

. The

interaction between peptides and CDs occurs through the hydrophobic amino acids and their

aromatic rings. The diameter of the βCD cavity allows the adjustment of residues phenylalanine,

tyrosine, histidine, and tryptophan in the CD hydrophobic cavity; however, interaction between

the peptides and αCD and γCD is less efficient because of steric interference 23, 24

.

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The aim of this study was to explore the antimicrobial activity and cytotoxicity of AMPs

prepared with βCD as well as the interactions of the peptides with the surface of periodontal

bacteria.

MATERIALS AND METHODS

Antimicrobial Peptides

LyeTxI was synthesized and purified by RP-HPLC as previously reported elsewhere 18

. LL37f

and KR12 were purchased from Anaspec**

. The peptide physicochemical properties are listed in

Table 1.

AMP/βCD Compound Preparation

A 1:1 AMP/βCD compound preparation was made using the freeze-drying method as previously

described 25

. Briefly, LyeTxI, LL37f, KR12, and βCD††

were dissolved separately in milli-Q®

water at a 1:1 molar ratio. The aqueous solution of each peptide was then mixture with the βCD

aqueous solution, stirred for 8 h, and then submitted to the freeze-drying process in order to

obtain a solid-associated compound.

Bacterial Susceptibility Assay

The susceptibility of periodontal bacteria to AMPs was assessed by determining the Minimum

Inhibitory Concentration (MIC) using the broth dilution method modified for the determination

of bacterial susceptibility to antimicrobial peptides as previously described 26, 27

. Briefly, a series

of two-fold dilutions of the AMPs and the AMP/βCD peptides were prepared in milli-Q® water

and settled in 96-well U-bottom polypropylene microtiter plates‡‡

. Fresh cultures of F. nucleatum

(ATCC 25586), P. gingivalis (ATCC 33277), and A. actinomycetemcomitans (ATCC 29522)

were diluted in an appropriate medium for each species and added to the microtiter plates which

were then incubated in the appropriated conditions according with the bacterial strain. The MIC

was defined as the lowest concentration that prevented visible growth of the microorganism.

Minimum Bactericidal Concentrations (MBCs) were determined by plating all the MIC wells,

which did not show any turbidity in supplemented Brain Heart Infusion (BHI) agar. After 24-48 h

of growth, the MBC was determined as the lowest concentration that did not permit visible

growth on the surface of the agar. All MIC and MBC assays were performed in triplicate.

Surface Charge Measurements

The influence of increasing concentrations (0-500 μg/mL) of the AMPs and AMPS/βCD on F.

nucleatum superficial charge was determined by zeta potential (ZP) measurements using a

Malvern Zetasizer Nano ZS§§

and the Laser Doppler Velocimetry technique. The experiment

was conducted at 25 ºC with a disposable cuvette (DPS1060). The reason of compared the

influence of AMPs and AMPs/βCD on F. nucleatum was made based on the results of the

antimicrobial efficacy of the pure AMPs and the βCD:AMPs, MIC and MBC.

AMPs and AMP/βCD stock solutions were prepared in 100 mM phosphate buffer, pH 7.4 at a

10-fold higher concentration than needed. Separately, a bacterial suspension at 108 CFU/mL was

prepared in 100 mM phosphate buffer, pH 7.4 from a fresh F. nucleatum culture. The solutions

used to suspend the bacteria and to prepare the AMP and AMP/βCD solutions were filtered (0.22

m filters***

) before use. Next, 100 μL of each standard solution of peptide or AMP/βCD

Journal of Periodontology; Copyright 2013 DOI: 10.1902/jop.2013.120679

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compounds were added to 900 μL of bacterial suspension. The suspension was transferred to a

disposable cuvette and the zeta potential measurement was made in triplicate.

Atomic Force Microscopy Imaging

A fresh culture of A. actinomycetemcomitans was adjusted at 108 CFU/mL in PBS and exposed

for 3 h to KR12 or KR12/βCD as representatives AMPs and AMP/βCD, respectively, to

determine the superficial roughness at the MIC concentration (250 and 16 μg/mL, respectively).

The βCD alone (60 μg/mL) was used as a blank control. After the exposure, the bacteria

suspension was washed twice with PBS and coated on a glass slide pre-treated with a HCl:HNO3

3:1 solution. The sample was air-dried at 25 ºC, fixed 1 h in 2.5% glutaraldehyde in 0.1 M

cacodylate buffer (pH 7.1) and dehydrated in graded alcohol. The bacteria images were acquired

in the equipment Asylum Research†††

.

Hemolytic Assays

The hemolytic activities of AMPs and the AMP/βCD compounds were determined by measuring

the hemoglobin release in suspensions of fresh rabbit erythrocytes at an absorbance of 414 nm as

previously described 28

in a 96-well U-bottom polypropylene microtiter plate. The AMPs and

AMP/βCD dilutions were made in PBS pH 7.4 and the tested concentrations were between 50

and 0.1 μg/mL.

Cytotoxicity Assays Using Osteoblast and Caco-2 Epithelial Cells

Osteoblasts were isolated from three-day-old Wistar rats calvaria using an enzymatic digestion

method previously described 29

. The osteoblasts were cultured in Dulbecco’s modified Eagle’s

medium (DMEM) ‡‡‡

supplemented with 10% fetal bovine serum (FBS) §§§

as well as antibiotic

(0.1 mg/mL streptomycin and 100 U/mL penicillin) and antimycotic solutions, and then

incubated at 37 oC in a humidified atmosphere of 95% air and 5% CO2. Once the cells reached

confluence, they were passaged and then used for the experiments.

Epithelial Caco-2 cells were obtained from the ATCC HTB-37 and used from passage 30 to

35. Cells were cultivated in DMEM-high glucose medium supplemented with 10% FBS, 3%

nonessential amino acids, 2 mM glutamine, 1% (vol/vol) HEPES, antibiotics (0.1 mg/mL

streptomycin and 100 U/mL penicillin), and antimycotics. The cells were cultured at 37 oC in a

humidified atmosphere of 95% air and 5% CO2.

After reaching confluence, each cell type was exposed 24 h to a series of two-fold dilutions of

AMPs and AMP/βCD compounds in the range of 100 to 0.78 μg/mL, Compound cytotoxicity

was assessed using an MTT assay as described by Mosmann 30

. The absorbance data were

converted to viability percentages based on the control, which contained culture medium only.

The viability percentages were compared by a two-way analysis of variance (ANOVA) test,

followed by a Bonferronni test, using the statistics software GraphPad Prism 5.0. The effective

concentration 50 (EC50) was defined as the value when the response is halfway between

minimum and maximum response in a dose-response curve 31

, which in this case was the

compound concentration required to achieve 50% cell death compared to the control. Pure AMPs

and AMP/βCD compounds EC50 from different cell types were determined by nonlinear

regression using the sigmoidal dose-response equation. The significance of the difference

between the EC50 was calculated using a Student’s t-test. Statistical significance was considered

at p < 0.05.

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RESULTS

AMPs and AMPs/βCD Antimicrobial Activity

The results of the susceptibility tests are shown in Table 2. The LyeTxI/βCD compound has

significantly greater antimicrobial activity than the pure peptide, against A.

actinomycetemcomitans (<0.03 vs. 30.38 μg/mL, respectively) and P. gingivalis (15.20 vs. 30.38

μg/mL, respectively). The activity of the LyeTxI/βCD compound against F. nucleatum showed

no change when compared to pure peptide.

The LL37f peptide had no antimicrobial activity at concentrations below 250 μg/mL.

However, the compound of LL37f with βCD had increased antimicrobial activity against the

bacteria tested. F. nucleatum exhibited the greatest sensitivity to KR12, with an MIC value of

125 μg/mL. We did not observe any antimicrobial activity for KR12 against A.

actinomycetemcomitans and P. gingivalis at concentrations below 250 μg/mL. However, the

KR12/βCD inclusion compound reduced significantly the MICs values against all bacteria tested.

Zeta Potential Measurements of the F. Nucleatum Surface

F. nucleatum exhibited a baseline negative Zeta potential of approximately -17 mV; however,

upon titration with increasing concentrations of LL37f, KR12, LL37f/βCD, and KR12/βCD (Fig.

1B and 1C), the surface Zeta potential increased to values of approximately -5 mV. Moreover,

titration with LyeTxI and LyeTxI/βCD caused the surface Zeta potential of F. nucleatum to

become positive by going through the isoelectric point (0 mV). This occurred at a lower

concentration for LyeTxI/βCD compared to pure peptide (~250 μg/mL and ~350 μg/mL,

respectively; Fig. 1A). Furthermore, the titration of increasing concentrations of βCD alone on F.

nucleatum (Fig. 1D) did not have a substantial effect on the zeta potential, which remained

approximately -10 mV at a concentration of 250 μg/mL of βCD.

AFM Imaging

AFM images of A. actinomycetemcomitans confirmed the typical shape of a small, round-ended

rod (length ± 1.20 μm, width ± 0.86 μm). After three hours of exposure to KR12 and KR12/βCD,

the surface roughness of A. actinomycetemcomitans markedly increased compared to the pure

βCD control (52.28 nm and 99.058 nm vs. 36.62 nm, respectively; Fig. 2B and 2C). To assess the

surface roughness changes was chosen A. actinomycetemcomitans based on the results founded in

the antimicrobial activity test (Table 2), which showed that there was moderate concentration to

MIC when treated with KR12/βCD against this bacterium. This approach eliminated the

possibility of extensive cell damage caused by higher compound concentration or longer

exposure time as observed with the other bacteria or peptides.

Hemolytic Activity of AMPs and AMP/βCD Compounds

As shown in Figure 3, the hemolysis generated by LL37f, KR12, and the respective peptides

associated with βCD was not more than 10% at 50 μg/mL. In contrast, LyeTxI and LyeTxI/βCD

generated 90% hemolysis at a concentration of 50 μg/mL. This result suggests that βCD/AMPs

inclusion compound in do not induce greater hemolysis compared to pure AMPs.

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Cytotoxicity of AMPs and AMP/βCD Compounds on Osteoblasts and Caco-2 Cells

We next assessed the cell viability of osteoblasts exposed to AMPs and AMP/βCD compounds

(Fig. 4). The average percent viability of cells treated with the AMPs or AMP/βCD compounds

were compared to a control that only contained culture medium. We found that treatment with 50

μg/mL of LyeTxl significantly reduced the cell viability of osteoblasts by 24.8 ± 2.9% (p < 0.05;

Fig. 4). The cytotoxicity of LyTxI/βCD was greater than pure LyeTxI, and significantly reduced

cell viability by 59.89 ± 14.88% at a dose of 25 µg/ml (p <0.05; Fig. 4). The LL37f and KR12

peptides were not cytotoxic to osteoblasts at a concentration less than 100 μg/mL, but the

LL37f/βCD and KR12/βCD preparations did induce a moderate reduction in cell viability at a

concentration of 100 µg/ml (57.61 ± 6.0% and 57.61 ± 6.0%, respectively; Fig. 4B and 4C).

When assessed the cytotoxic effects of the AMPs and AMP/βCD compounds on Caco-2

epithelial cells both LyeTxI and LyeTxI/βCD were significantly cytotoxic at a concentration of

6.25 μg/mL. LL37f and LL37f/βCD exhibited cytotoxic effects against the cells at a

concentration of 50 and 25 μg/mL, respectively. In addition, KR12 decreased cell viability at a

concentration of 25 μg/mL, and the KR12/βCD compound exhibited an even greater cytotoxic

effect by reducing cell viability at a concentration of 12.5 μg/mL (p < 0.05).

Table 3 lists the EC50 values of the AMPs and AMPs/βCD compounds in osteoblasts and

Caco-2 cells. The EC50 values of the peptides and compounds with βCD were greater than those

required to achieve inhibition of bacterial growth in all species tested. Importantly, the cytotoxic

concentrations of the AMPs and AMPs/βCD were lower for epithelial cells than for osteoblasts.

However, βCD alone did not induce a statistically significant decrease in cell viability for both

cell types compared to the blank control (p < 0.05; Fig. 4D and 5D).

DISCUSSION

It has been previously shown that periodontal bacteria are susceptible to AMPs in vitro 26, 32

;

however, the concentration needed to obtain an inhibitory effect was as high as 200 μg/mL. This

concentration may be considered too high for the practical use of AMPs in the treatment of

periodontal disease, which makes the production of pharmaceutical formulations a very

expensive process. In the present study, we explored the AMPs included in βCD as a strategy to

circumvent this limitation, since the inclusion of peptides on CDs has been shown to improve

solubility and stability, which could be the result of stabilization of the functional form of the

native protein in solution 21, 22

. In addition molecular inclusion plays an important role in the

reduction of the antimicrobial concentration required for inhibitory activity 33 - 36

.

Furthermore, AMPs and AMP/βCD compounds exhibited an additional influence on the

surface roughness and bacterial cell surface charge properties based on zeta potential

measurements. Bacterial surface root–mean–square roughness (Rrms), after incubation with KR12

or KR12/βCD at the MIC concentration, significantly increased when compared to the βCD

control (Fig. 2). Previous reports have found that the bacterial surface Rrms increases after AMP

treatment and is dependent on the antibiotic concentration and incubation time 37-40

. The use of

low concentrations of compounds allowed us to observe the initial morphological changes, such

as surface indentations, micelle-like structures, and outer membrane residues. Thus, outer

membrane disruption and the release of LPS molecules from the bacteria surface may be the

reason for the increase in surface roughness41

. These results also validate our previous report of

nanoaggregates formation on the bacterial surface after treatment with an inclusion compound42

.

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The initial Zeta potential measurements of F. nucleatum in our study were approximately -17

mV due to LPS on the cell surface. As the concentration of AMP and AMP/βCD increased, the

surface charge increased, and in some cases, came close to zero or became positive. This may be

due to membrane destabilization induced by the AMPs and AMPs/βCD compounds. Importantly,

the surface zeta potential of F. nucleatum did not increase with increasing concentrations of βCD

even at high concentrations (250 µg/ml), indicating that the bacterial surface electrostatic changes

observed after exposure to the AMP/βCD compounds were not due to the presence of βCD alone.

The increased interaction of the AMP/βCD compounds may be caused by adhesion of βCD to the

bacterial surface through hydrogen bonds, which could act synergistically with the ionic

interactions between the cationic peptide and the anionic cell surface.

On the other hand, the interaction differences with cell membranes between the AMPs and

AMP/βCD compounds tested could be explained by differences in their total charge and

hydrophobicity (Table 1), since balancing peptide hydrophobicity and charge distribution

promotes efficient antimicrobial activity 43

. LyeTxI has a more positive charge than the others

peptides (+5), which may be why this peptide was the only one capable of reversing the surface

charge of F. nucleatum. In biological systems, the neutralization of surface charge can be largely

attributed to the balance of electrostatic interactions between the positively charged peptide (side

chains of lysine and arginine) and the negatively charged groups (phosphates and carboxylates)

of LPS 44

.

Our assessment of the AMP and AMP/βCD cytotoxic potential found statistical differences

between the EC50 values compounds on Caco-2 cells, which let us to conclude that βCD increases

the cellular toxicity of these peptides against the cells (Fig. 5). This has important implications on

the treatment of periodontal disease, since epithelial proliferation after surgical treatment does not

allow efficient epithelial regeneration 9, 10

. AMPs and AMP/βCD compounds were not cytotoxic

against osteoblasts, and were not hemolytic at the bactericidal concentrations used against the

tested bacteria (Figs. 3 and 4). The low cytotoxicity of the AMPs in mammalian cells compared

to the cytotoxicity potential against bacteria could be explained by the fact that bacterial

membranes contain a greater amount of negatively charged phospholipids, which allows for a

stronger interaction between cationic peptides and the membrane 45

. In contrast, the outer

membrane of the phospholipids bilayer of normal mammalian cells is composed predominantly

of zwitterionic phospholipids (e.g. phosphatidylcholine and sphingomyelin) 46,

47

.

In this study, we found that LyeTxI and LyeTxI/βCD were the most hemolytic compounds.

Some α-helical AMPs have a tendency to be highly hemolytic 48

, and recent studies have shown

that high hydrophobicity as well as high α-helicity or a β-sheet structure were correlated with

increased toxicity, as measured by hemolytic activity 49

. To be useful as a broad spectrum

antibiotic, it would be necessary to change the ratio between the anti-eukaryotic activity and the

antimicrobial activity, which could be accomplished using several strategies, such as increasing

antimicrobial activity, decreasing hemolytic activity while maintaining antimicrobial activity.

In conclusion, LyeTxI/βCD, LL37f/βCD, and KR12/βCD inclusion compound are effective

as antimicrobial agents by the capacity to inhibit periodontal pathogens such as: A.

actinomycetemcomitans, F. nucleatum, and P. gingivalis at a low concentration, as well as, the

epithelial cells proliferation at non-cytotoxic doses for osteoblasts or erythrocytes.

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8

ACKNOWLEDGEMENTS

This work was supported by the Brazilian funding agencies, INCT-Nanobiofar, CNPq, CAPES, FAPEMIG, and

INCT-TOX-FAPESP. We thank Luciana Moreira from the Microscopy Center – UFMG for her important help in the

AFM images acquisition.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

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§ Restorative Dentistry Department, Dentistry Faculty, Federal University of Minas Gerais,

UFMG, Av. Antônio Carlos 6627, 31270-901. Belo Horizonte – MG, Brazil. (Phone: +55-31-

3409-2430 Fax: +55-31-3409-5700) mecortes@ufmg.br (This could be published)

Submitted November 15, 2012; accepted for publication February 26, 2013.

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Figure 1.

Influence of increasing concentrations of the AMPs and AMPS/βCD on F. nucleatum (ATCC 25586) superficial

charge determined by zeta potential measurements using a Malvern Zetasizer Nano ZS and the Laser Dopple

Velocimetry technique. (A) LyeTxI vs. LyeTxI/βCD titration. (B) LL37f vs. LL37f/βCD titration. (C) KR12 vs.

KR12/βCD titration (D) βCD Titration. 145x301mm (300 x 300 DPI)

Figure 2.

Surface roughness of A. actinomycetemcomitans surface by Atomic Force Microscopy after exposure with (A) βCD

(B) KR12; (C) KR12/βCD. 280x391mm (96 x 96 DPI)

Figure 3.

Hemolysis percent of rabbit erythrocytes generated by increasing concentrations of LyeTxI, LL-37f KR-12 and their

compounds associated with βCD. 126x69mm (300 x 300 DPI)

Figure 4.

Osteoblasts cytotoxicity test with increasing concentrations of the AMPs and AMPs/βCD. (A) LyeTxI and

LyeTxI/βCD; (B) LL37f and LL37f/βCD;(C) KR12 and KR12/βCD, (D) βCD. 789x437mm (96 x 96 DPI)

Figure 5.

Caco-2 Epithelial cells cytotoxicity test with increasing concentrations of the AMPs and AMPs/βCD. (A) LyeTxI and

LyeTxI/βCD; (B) LL37f and LL37f/βCD;(C) KR12 and KR12/βCD, (D) βCD 745x450mm (96 x 96 DPI)

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Table 1.

LyeTxI, LL37f e KR12 peptides physico-chemical properties

Peptide/Sequence Total Charge Hydrophobic

residues (%)

LL37f

KRIVQRIKDFLRNLVPRTES +4 35

KR12

KRIVQRIKDFLR +4 41

LyeTxI

IWLTALKFLGKNLGKHLAKQQLAKL +5 52

Table 2.

Periodontopathogenic bacteria susceptibility to AMPs and their compounds associated with βCD. Values

expressed as μg/mL.

Compound

Aa Fn Pg

MIC MBC MIC MBC MIC MBC

LyeTx I 30.38 60.75 30.38 30.38 30.38 30.38

LyeTx I/βCD 1:1 <0.03 60.75 30.38 30.38 15.20 60.75

LL37f >250 >250 250 >250 250 >250

LL37f /βCD 1:1 31.25 >250 62.5 125 7.81 62.5

KR-12 >250 >250 125 125 >250 >250

KR-12/βCD 1:1 15.63 31.25 7.81 7.81 62.5 250

Aa: Aggregatibacter actinomycetemcomitans

Fn: Fusobacterium nucleatum

Pg: Porphyromonas gingivalis

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Table 3.

AMPs and AMPs/βCD effective concentration 50 (EC50) in osteoblasts and epithelial Caco-2 cells.

Compound EC50 (μg/mL)

Osteoblast p Value

EC50 (μg/mL)

Caco-2 Cells p Value

LyeTxI 36.98 >0.05

99.8 <0.05

LyeTxI/βCD 37.16 9.21

KR12 688.0 <0.05

163.1 >0.05

KR12/βCD 168.0 104.5

LL37f 990.2 <0.05

227.2 <0.05

LL37f/βCD 119.5 96.04

** Eurogenetec Group, California – USA

†† Xiamen Mchem Pharma, China

‡‡ Costar® # 3879, Corning USA

§§ Malvern Instruments, Malvern, UK

*** Millipore

††† Santa Barbara, CA, USA

‡‡‡ Sigma, St Louis, USA

§§§ Gibco, NY, USA

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