Universidade de Lisboa

Faculdade de Farmácia

Antifouling strategies to prevent catheters-

associated medical infections

Sara Manuela Costa Lemos

Mestrado Integrado em Ciências Farmacêuticas

2017

Universidade de Lisboa

Faculdade de Farmácia

Antifouling strategies to prevent catheters-

associated medical infections

Sara Manuela Costa Lemos

Monografia de Mestrado Integrado em Ciências Farmacêuticas apresentada à

Universidade de Lisboa através da Faculdade de Farmácia

Orientadora: Doutora Isabel Alexandra Caldeira Ribeiro, Professora

Auxiliar

2017

4

5

Resumo

O uso de dispositivos médicos invasivos tem vindo a aumentar com o passar dos anos,

sendo que os cateteres são dos dispositivos médicos mais utilizados.

Contudo há um risco de desenvolvimento de infeções associado ao uso destes

dispositivos, uma vez que os cateteres são feitos de materiais que, devido às suas superfícies

hidrofóbicas, são muito propensos à adesão de microrganismos. Deste modo, a prevenção da

adesão bacteriana à superfície destes materiais reveste-se de grande importância.

Este trabalho faz uma revisão de algumas estratégias anti-adesivas obtidas através da

modificação das propriedades físico-químicas dos materiais. Estratégias tais como os

revestimentos com polietilenoglicol, zwitteriões, polissacáridos, poliacrilamida e poliacrilatos,

polímeros anfifílicos e poliuretanos modificados foram abordadas. Também a Topografia de

Sharklet e as Superfícies Porosas Escorregadias Infundidas em Líquido (SLIPS) foram alvo de

revisão.

Apesar de todas as estratégias abordadas terem demonstrado ser eficazes na prevenção

da adesão bacteriana, o uso de zwitteriões (mais especificamente SBSi, PDA/PMEN10 e Poly-

SB), escovas Amino-PPX-PAAm, metilcelulose e a associação de diferentes polissacáridos

(nomeadamente a heparina com o quitosano ou a agarose) para o revestimento das superfícies

dos biomateriais são alvo de destaque. Também as SLIPS e a Topografia de Sharklet

demonstraram ser candidatos promissores na área das estratégias anti-adesivas.

Quanto à aplicação deste tipo de estratégias em cateteres, será necessária a realização

de mais estudos in vivo e ensaios clínicos em humanos para que se possa garantir a segurança

do uso destas estratégias.

Palavras-chave: Cateter; Adesão bacteriana; Biofilme; Infeção; Anti-adesivo;

6

Abstract

The use of invasive medical devices is becoming more common in nowadays, with

catheters representing one of the most used medical devices. However, there is a risk of

infection associated with the use of these devices, once they are made of materials that are prone

to bacterial adhesion. Therefore, to avoid the nefarious consequences of these infections the

prevention of bacterial adherence to the surface of catheters is an important aspect.

This review is focused in some strategies that are able to modify the physical or

chemical properties of materials, leading to the creation of antiadhesive surfaces. Strategies

such as coating the surfaces with poly(ethylene glycol), zwitterions, polysaccharides,

polyacrylamide and polyacrylates, amphiphilic polymers and modified polyurethanes were

reviewed. Also, two quite different approaches were included, the Sharklet topography and the

Slippery Liquid-Infused Porous Surfaces.

All the reviewed strategies have proven to be effective in preventing bacterial adhesion.

However, the best examples were the use of zwitterionic polymers (more specifically SBSi,

PDA/PMEN10 and Poly-SB), Amino-PPX-PAAm brushes, methylcellulose grafting and

associations of polysaccharides (heparin with chitosan or agarose) on biomaterials coatings.

Also, SLIPS and Sharklet Topography seem to be promising candidates in the field of

antifouling strategies.

Concerning the potential application of most of these strategies in catheters, more in

vitro studies and clinical trials in humans are needed, to assure the safety in possible future use.

Keywords: Catheter; Bacterial adhesion; Biofilm; Infection; Antifouling;

7

Acknowledgements

To my parents who always believed in me, even when I doubted of myself. For all the times

that they didn’t let me give up, for the strength they have always given me and for the pillar

that they are in my life.

To my family that always supported me and that always showed the pride that has for me. For

all the words of comfort and affection.

To the friends I did in college. For all the motivation they have always given me, for all the

moments we shared and for the certainty that they will be for life.

To those long-time friends who have seen me grow as a person and have been witnessing my

achievements.

To my friend João Pedro, for all the help, the advices, support and patience.

And finally, last but by no means least, to my advisor Dr. Isabel Alexandra Caldeira Ribeiro

for the continuous support during the writing process of my thesis and related research, for her

patience, guidance and immense knowledge.

To all, thanks so much for the encouragement.

8

Abbreviations and Acronyms

HAIs Healthcare-Associated Infections

ECDC European Centre for Disease Prevention and Control

EPS Extracellular Polymeric Substances

PD Peritoneal Dialysis

PVC Polyvinylchloride

PU Polyurethane

PDMS Polydimethylsiloxane

PTFE Polytetrafluoroethylene

PEG Poly(ethylene glycol)

CFUs Colony Forming Units

SEM Scanning Electron Microscopy

PPG Poly(propylene glycol)

OA Octadecylacrylate

Poly(PEGA/OA) Poly(ethylene glycol) Acrylate Copolymer

Poly(PEG-SO3A/OA) Sulfonated Poly(ethylene glycol) Acrylate Copolymer

PDA Polydopamine

PEO Poly(ethylene oxide)

SPU Segmented Polyurethane

PTMO Poly(tetramethylene oxide)

DS Dermatan Sulfate

Hep Heparin

MeCe Methylcellulose

TIVAP Totally Implantable Venous Access Port

AG Agarose

9

CH Chitosan

CMCS Carboxymethyl Chitosan

CAUTI Catheter-Associated Urinary Tract Infection

PCU Polycarbonate-Urethane

PIME Diamino-diamide-diol

MTT 3-(4,5) dimethylthiazol-2-yl-2,5 diphenyl tetrazolium bromide

UV Ultraviolet

MSNs Mesoporous Silica Nanoparticles

AMSNs Agarose-Loaded Mesoporous Silica Nanoparticles

AHMSNs Agarose and Heparin-Loaded Mesoporous Silica Nanoparticles

PUh Hexamethylene Diisocyanate Based Polyurethanes

HDI Hexamethylene Diisocyanate

PAAm Polyacrylamide

Amino-PPX Amino-poly(o-amino-p-xylylene-co-p-xylylene)

ATRP Atom Transfer Radical Polymerization

PA13 Poly(methylmethacrylate-co-dimethylacrylamide)

PA515 Poly(methoxyethylmethacrylate-co-diethylaminoethylacrylate-

-co-methylmethacrylate)

PA155 Poly(hydroxyethylmethacrylate-co-dimethylaminoethylme-

thacrylate)

SB Sulfobetaine

CB Carboxybetaine

PC Phosphorylcholine

Poly-SB Polymeric Sulfobetaine

SBMA Sulfobetaine Methacrylate

SBSi Sulfobetaine Silane

PMENs Copolymers Bearing Phosphorylcholine and Active Ester Groups

10

MPC 2-Methacryloyloxyethyl Phosphorylcholine

NPCEMA p-Nitrophenoxycarbonyloxyethyl Methacrylate

P4VP Polymer 4-vinyl Pyridine

PCB Protected Carboxybetaine

DMA Dodecyl Methacrylate

PEGMA Poly(ethylene glycol) Methacrylate

AA Acrylic Acid

PBS Phosphate Buffered Saline

O.D. Optical Density

EA Ethanolamine

S Serinol

ED Ethylene Diamine

MDI Methylene-bis-phenyl-isocyanate

DHMPA Dihydroxymethyl-propionic Acid

PLC Polycaprolactide

PLA Poly-l-lactide

PPO Polypropylenoxide

SLIPS Slippery Liquid-Infused Porous Surfaces

TLP Tethered Liquid Perfluorocarbon

PET Polyethylene Terephthalate

LSM Confocal Laser Scanning Microscopy

UTI Urinary Tract Infection

11

Index:

1 Introduction ...................................................................................................................... 13 2 Materials and methods ..................................................................................................... 16 3 Catheters and their materials ............................................................................................ 17 4 Antifouling strategies ....................................................................................................... 19

4.1 Hydrophilic polymers ............................................................................................... 19 4.1.1 Poly(ethylene glycol) and derivates ..................................................................... 19 4.1.2 Polysaccharides .................................................................................................... 23

4.1.2.1 Dermatan Sulfate .......................................................................................... 23 4.1.2.2 Methylcellulose ............................................................................................ 24

4.1.2.3 Agarose ......................................................................................................... 25 4.1.2.4 Chitosan ........................................................................................................ 25 4.1.2.5 Heparin ......................................................................................................... 26

4.1.3 Polyacrylamide and polyacrylates ........................................................................ 31 4.2 Zwitterionic Polymers .............................................................................................. 34

4.2.1 Betaines ................................................................................................................ 34 4.3 Other antifouling coatings ........................................................................................ 38

4.3.1 Amphiphilic polymers .......................................................................................... 38 4.3.2 Modified Polyurethanes ....................................................................................... 39 4.3.3 Slippery Liquid-Infused Porous Surfaces ............................................................ 40 4.3.4 Sharklet topography ............................................................................................. 42

5 Discussion ........................................................................................................................ 46 6 Conclusion ........................................................................................................................ 48

Bibliography ............................................................................................................................. 49

Index of Figures:

Figure 1 - Steps of biofilm formation ....................................................................................... 14

Figure 2 - Representation of an antifouling surface ................................................................. 15

Figure 3 - Representation of a SLIPS surface ………………………………………………... 41

Index of Tables:

Table 1 – Types of catheters, their materials and the most common microorganisms related with

the development of infection ............................................................................................ 18

Table 2 – Use of PEG and derivates as antifouling strategies with possible application in

catheters ............................................................................................................................ 22

Table 3 – Use of polysaccharides as antifouling strategies with possible application in catheters

.......................................................................................................................................... 30 Table 4 – Use of PAAm and polyacrylates as antifouling strategies with possible application in

catheters ............................................................................................................................ 33 Table 5 – Use of zwitterions as antifouling strategies with possible application in catheters . 37

Table 6 – Use of amphiphilic polymers as antifouling strategies with possible application in

catheters ............................................................................................................................ 44 Table 7 – Use of modified PUs as antifouling strategies with possible application in catheters

.......................................................................................................................................... 44 Table 8 – Use of SLIPS as antifouling strategies with possible application in catheters ........ 45

12

Table 9 – Use of Sharklet Topography as antifouling strategy with possible application in

catheters ............................................................................................................................ 45

13

1 Introduction

Invasive medical devices are becoming more important each day on medical modern

practice since they can improve therapeutic results and save many lives. However, these type

of devices also represents an important risk factor for the development of healthcare-associated

infections (HAIs), particularly in more susceptible patients (1,2). HAIs, also known as

“nosocomial” or “hospital infections”, are infections that patients acquire while they are

receiving a treatment for a previous illness in a healthcare facility. HAIs are the most common

adverse events in healthcare facilities worldwide, with an important impact on morbidity,

mortality and quality of life, and also representing significant financial expenses to the health

systems (3). According to the 2014 annual epidemiological report of European Centre for

Disease Prevention and Control (ECDC), the prevalence of patients with at least one HAI in

European acute‑care hospitals in 2013 was estimated at 3.4%, with a total of 4.2 million HAIs

in the entire year (4).

Currently, one of the most important devices used in medical procedures that are related

to HAIs are catheters (5). In fact, in 2013, 43.3% of bloodstream infections were reported to be

catheter-related and 96.7% of urinary tract infections were associated with the use of urinary

catheters in UCI (4). Some of the most frequent agents of these type of infections are bacteria,

but can also be parasites, fungi, viruses and prions (6).

Vascular and urinary catheters are currently the most used invasive medical devices

(2,7). However, on catheters, bacteria (and sometimes other microorganisms and cells) have

the ability to adhere and promote the formation of a biofilm on the surface of these devices

(1,7,8).

Biofilms are communities of either homogeneous or heterogeneous populations of

bacteria that can strongly attach to both biotic and abiotic surfaces, and self-produce a matrix

made up of extracellular polymeric substances (EPS), that basically consists of polysaccharides,

proteins, nucleic acids, lipids and other materials. The EPS acts as a scaffold that holds the

biofilm together, stabilizing the biofilm network and protecting the bacteria from external

threats (6,9,10). Biofilm formation includes several stages: reversible and irreversible

attachment, colonization, maturation and dispersion (Figure 1) (6,11). In the first stage – the

attachment – planktonic bacteria adhere to the surface mainly by weak physical forces such as

electrostatic interactions or van der Waals forces, or by some bacterial appendages like flagella,

pili and fimbriae, that results in a reversible attachment. However, with the time, bacterial

14

appendages overcome the physical repulsive forces, making bacterial cells irreversibly attached

(6,9,12). In the next stage – the colonization and maturation of biofilm – bacteria already

adhered to the surface start producing autoinducer signals that allow cell-to-cell communication

and start building a matrix of EPS. In this stage biofilm comes multilayered, the bacterial colony

grows and its density increases (9). Then, due to changes in nutrients, temperature and oxygen

levels at the latter end of the biofilm cycle, cells that were part of a complex, relatively static,

slow-growing community within the biofilm become differentiated and often highly motile

microorganisms. Parts of biofilm detach within the host and the dispersed cells are able to cause

systemic infection, particularly in immunocompromised patients, since they can attach to new

surfaces and colonize new sites (6).

The presence of a matrix in biofilms is an important obstacle because it protects bacteria

from the innate immune defenses of the host, difficults antimicrobials to penetrate the full depth

of biofilm conferring tolerance to antibiotic treatments, and allows a better cell-to-cell

communication with transference of some drug-resistance markers and virulence factors,

resulting in a long-term persistence of these microorganisms in medical devices (9,10,13).

Due to the difficulty of eradication biofilms from the surfaces of catheters, the removal

of the contaminated catheter is frequently the only solution to treat infections. That is why the

prevention of biofilm formation in these devices is so important in the reduction of HAIs. This

prevention may be achieved by different strategies such as: 1) coating the surface of medical

devices with biocidal agents; 2) modifying the physicochemical properties of polymers creating

antifouling surfaces; 3) associating both antifouling and bactericidal properties (1,14,15). In

this review, the antifouling strategies will be emphasized. Many clinical trials show results of

Attachment Colonization Maturation Dispersion

Figure 1 – Steps of biofilm formation

15

a possible increase antibiotic resistance in long-term use of biocidal coatings therefore

antifouling strategies are an interesting choice in fighting biofilms. These strategies consist in

physicochemical surface modifications focusing on roughness, nanostructures and

hydrophobicity of the biomaterials, resulting in antiadhesive materials that prevent bacterial

attachment and consequently biofilm formation on medical devices (Figure 2) (14,16).

The aim of this work is to review antifouling strategies with possible application on

biomedical devices. This review will be focused on catheters in order to prevent microbial

attachment and proliferation with consequent biofilm formation on the surface of these medical

devices.

Bare catheter Coated catheter

Figure 2 – Representation of an antifouling surface

16

2 Materials and methods

Online databases containing medical, technical and scientific contents (i.e. Web of

Knowledge, PubMed, Science Direct and Springer) were used to the development of this work.

For the epidemiological data, 2014 Annual epidemiological report of the European Centre for

Disease Prevention and Control (ECDC) was used.

The first stage of the research was made by using some general keywords like

“nosocomial infections”, “bacteria”, “catheters”, “antifouling”, “antiadhesive”, “medical

devices”, “antibiofilm” and “antiadhesive polymers”. Review articles and book sections were

the materials of choice to this first phase.

After that, a more specific research was made. In this stage combinations of specific

keywords such as “sulfobetaine catheter”, “polysaccharide catheter” and “PEG medical

devices” were used. Although there some articles from before 2009 were referenced,

bibliography sources from the last 7 years were privileged. Studies conducted specifically on

catheters and also on catheters-materials were considered.

17

3 Catheters and their materials

The ideal catheter should have some characteristics such as high tensile strength,

biocompatibility, be soft and pliable and inherently chemical-resistant. It is a fact that these

properties are conferred by the materials of these devices (17,18).

There are three principal types of catheters: vascular, urinary and peritoneal dialysis

(PD) catheters. However, the most commonly used ones are the first two (1,2).

Urinary catheters are inserted through the urethra into the bladder, with the purpose to

collect urine during surgical procedures, measure urine output or control urinary incontinence

or retention (6,9). They are made of materials such as gum-elastic, polyvinylchloride (PVC),

polyurethane (PU), silicone and latex. Among these silicon, more specifically

polydimethylsiloxane (PDMS) (a type of silicone), is the most used material (18,19).

The most common agents that can colonize urinary catheters are, for example, some

Gram-positive bacteria such as Staphylococcus epidermidis, and some Gram-negative bacteria

such as Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae (9,20).

Vascular catheters may be used to administer fluids, medication or nutrients or to make

dialysis. The most common materials of this medical devices are PU, silicone, polyethylene and

polytetrafluoroethylene (PTFE) (also known as Teflon®). However, PU is the most used one,

due to it high biocompatibility, resistance to several chemicals and compatibility with many

drugs, and also because it is thromboresistant and becomes less rigid inside the body, reducing

mechanical trauma and vein irritation. S. epidermidis, S. aureus, Candida albicans and

Pseudomonas aeruginosa are some of the main reported pathogens responsible for HAI’s

associated to these catheters (6,9,17,21).

PD is a therapy used in some cases of severe renal disease, that delay the degradation

of the residual renal function, at the same time that improves quality of life of patients and

maintains the hemodynamics (22,23). For this technique, a PD catheter (mainly constituted by

silicone) is inserted into the abdominal cavity of the patient, and a dialysis solution is introduced

into this cavity, and subsequently removed. The peritoneum acts like a semi-permeable

membrane (1,24,25). This type of catheters is also suitable to bacterial colonization. In fact, at

least 75% of peritonitis are related to colonization of Gram-positive bacteria such as S.

epidermidis and S. aureus, followed by Gram-negative bacteria colonization including P.

aeruginosa in approximately 20% of the cases (25,26).

18

The principal types of catheters, their materials and the most common microorganisms

that are related with the development of infection are summarized in Table 1.

Table 1 – Types of catheters, their materials and the most common microorganisms

related with the development of infection

Common materials Most common pathogens Ref.

Urinary

Catheter Gum-elastic, PVC, PUs,

silicone, PDMS and latex.

Some Gram-positive bacteria (S. epidermidis), and some

Gram-negative bacteria such as E. coli, P. mirabilis and

K. pneumoniae.

(9,18–20)

Vascular

Catheter PU, silicone, polyethylene

and PTFE (Teflon®). S. epidermidis, S. aureus, C. albicans and P. aeruginosa. (6,9,17,21)

PD Catheter Mainly silicone.

Mostly Gram-positive bacteria (S. epidermidis and S.

aureus) followed by Gram-negative bacteria (P.

aeruginosa)

(1,24–26)

19

4 Antifouling strategies

As previously mentioned, the first step on catheter-associated medical infections is the

attachment of bacteria to the surface of this medical devices, since both catheter and the

majority of bacteria’s surfaces are hydrophobic (which increase the strength of hydrophobic

interactions) (1,8,14,27,28). With the purpose of preventing that initial attachment, antifouling

polymers have been developed. These polymers have the capacity to resist to bacterial

attachment by one of the following mechanisms: increasing surface hydrophilicity, adding

negative charges or decreasing surface free energy (27,29,30).

There are two principal types of antifouling polymers, depending on how they interact

with water molecules: hydrophilic polymers and zwitterionic polymers (18,31). These polymers

create a water layer on the surface of biomaterials. When bacteria or other foulants such as

proteins get closer to the surface, the polymer chains are compressed and the water molecules

previously bonded to polymer coatings are released (1,32,33). These two events leads to a

decrease in entropy and increase in enthalpy, respectively, which results in a

thermodynamically unfavorable surface that repels bacteria (34).

Thus, antifouling polymers can avoid the development of infections on medical devices

such as catheters (8).

4.1 Hydrophilic polymers

Hydrophilic polymers are polymers that have chemical affinity to water molecules (35).

These polymers have the ability to bond with the surrounding water by hydrogen bonds creating

a hydrated layer on their surface (31,36). However, since these bonds are relatively weak,

changes in surface hydration can occur with the time, which can lead to loss of antiadhesive

properties of these materials (35).

4.1.1 Poly(ethylene glycol) and derivates

Poly(ethylene glycol) (PEG) is a neutral and hydrophilic polymer that has been widely

studied relatively to its antiadhesive properties (1,36–39). The ether groups of this polyether

are able to bond with several water molecules through weak hydrogen bonds, forming a

hydrated layer on biomaterials surfaces (29,37,40).

20

There are many different strategies to attach PEG to the surface of biomaterials.

Covalent grafting, chemical or physical adsorption, plasma deposition and graft or block

copolymerization are some of the options (29,38,41).

Through the last decades many studies have been developed aiming to evaluate the

efficacy of PEG coatings on the prevention of bacterial colonization on the surface of medical

devices.

One of those studies was developed by Park et al. (42) who synthetized different PEG

chains with the same length but different terminal groups (PEG1k-OH, PEG1k-NH2 and

PEG1k-SO3H), and a PEG chain with a higher length (PEG3.4k-OH) (Table 2). The synthetized

PEG chains were grafted on PU beads and the number of adhered E. coli (ATCC 11775) and

S. epidermidis (ATCC 12228) cells was quantified by the number of Colony Forming Units

(CFUs) per cm2 and surface observed through scanning electron microscopy (SEM).

Comparing both controls, the bare PU and the hydrophobic poly(propylene glycol) (PPG)

grafted PU surfaces (PPG1k-OH), with PEG-1k coatings, the authors concluded that S.

epidermidis and E. coli adhesion in plasma reduced. Also, both PEG1k-SO3H and PEG3.4k

surfaces showed the best antifouling results for the two types of bacteria (42).

In another study, H. Lee et al. (43) developed and characterized new PEG and

sulfonated PEG acrylate copolymers containing a hydrophilic portion of PEG/PEG-SO3

acrylate and a hydrophobic portion of octadecylacrylate (OA). The obtained copolymers were

named as poly(PEGA/OA) and poly(PEG-SO3A/OA), respectively. The authors synthetized

different PEG/PEG-SO3 containing acrylate copolymers with three different chain lengths (1K,

2K and 4K). The obtained copolymers (Poly(PEG1KA/OA), Poly(PEG2KA/OA),

Poly(PEG4KA/OA), Poly(PEG1K-SO3A/OA), Poly(PEG2K-SO3A/OA), and Poly(PEG4K-

SO3A/OA)) were grafted onto PU films. The adhesion of S. epidermidis (KCTC 1917) and E.

coli (KCTC 2441) was assessed by SEM observation and determination of CFUs/cm2. The

obtained data demonstrated that PEG/PEG-SO3 acrylate copolymers were more effective in

repealing E. coli than S. epidermidis. It was also concluded that longer chain lengths of PEG

revealed a higher reduction on S. epidermidis adhesion, with reductions of ≈ 95%.

Comparatively to poly(PEGA/OA), poly(PEG-SO3A/OA) copolymers demonstrated lower S.

epidermidis attachment in broth, while E. coli reduction was observed on broth and plasma

(Table 2) (43).

21

More recently, in 2017, C. Xing et al. (44) immobilized PEG-OH and PEG-COOH on

polydopamine (PDA) pre-coated silicon wafers, obtaining the polymers PDA/PEG-OH and

PDA/PEG-COOH, respectively. To test the antibiofilm activity the authors used E. coli, S.

aureus and P. aeruginosa suspensions in the bacterial adhesion tests (Table 2). After 7 days,

the obtained results from fluorescence microscopy demonstrated that both PEG coatings

presented a significant reduction on bacterial attachment of the three tested strains. However

PDA/PEG-OH was the coating that demonstrated the best antifouling results (44).

The antifouling properties of a derivate of PEG was also evaluated. One of those studies

was performed by J. Park et al. (45) that developed different poly(ethylene oxide) (PEO)-based

multiblock copolymer/segmented PU (SPU) blends (Table 2). The multiblock copolymer was

composed by an hydrophilic portion of PEO and an hydrophobic portion of poly(tetramethylene

oxide) (PTMO) blocks. For this study the authors added 5, 10, 20 or 30 wt% of copolymer to

the SPU, and obtained the copolymers Pell-5, Pell-10, Pell-20 and Pell-30. From the bacterial

tests the authors concluded that Pell-5 was sufficient to significantly decrease the number of

adherent bacteria on the surfaces (83 ± 13 x 102 CFU/cm2 versus 275 ± 31 x 102 CFU/cm2 on

bare SPU). They also concluded that increasing the amount of copolymer on blends, the

resistance to bacterial attachment was higher. In fact, Pell-30 was the blend with less bacterial

adhesion of all the four blends tested. All four copolymers demonstrated be more effective

inhibiting S. epidermidis adhesion than P. mirabilis adhesion (45).

Although PEG is a very used polymer to confer antiadhesive properties to biomaterials,

it is easily oxidized in the presence of O2, forming aldehydes and ethers. This reaction results

in the degradation of PEG with a consequent decrease of it antifouling ability. For this reason,

PEG-coatings have a limited utility in long-term applications (35,36,38,46).

22

Table 2 – Use of PEG and derivates as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Uri

na

ry PEO/PTMO

multiblock

copolymer/

segmented

polyurethane blends

Polyurethane Pell-5; Pell-10;

Pell-20; Pell-30 Number of CFUs/cm2

E. coli (ATCC

11775), S.

epidermidis (ATCC

12228) and P.

mirabilis (ATCC

25993)

Pell-30 demonstrated < 50 x 102 CFUs/cm2 of E. coli,

P. mirabilis and S. epidermidis (versus 275 x 102

CFUs/cm2, ≈ 200 x 102 CFUs/cm2 for and ≈ 12000 x

102 CFUs/cm2 on controls, respectively).

(45)

Va

scu

lar

PEG grafting Polyurethane

PPG1k-OH; PEG1k-OH;

PEG1k-NH2; PEG1k-

SO3H; PEG3.4k-OH

Number of CFUs/cm2;

SEM

S. epidermidis

(ATCC 12228) and

E. coli (ATCC

11775)

≥ 90% reduction of the number of CFUs/cm2 on PU-

PEG1K-SO3H and PU-PEG3.4K-OH in plasma. (42)

Sulfonated PEG-

acrylate copolymer

surfaces

Polyurethane

Poly(PEG1KA/OA)

Poly(PEG2KA/OA)

Poly(PEG4KA/OA)

Poly(PEG1K-SO3A/OA)

Poly(PEG2K-SO3A/OA)

Poly(PEG4K-SO3A/OA)

Number of CFUs/cm2;

SEM

S. epidermidis

(KCTC 1917) and E.

coli (KCTC 2441)

> 80% reduction on S. epidermidis and > 90% on E.

coli adhesion on Poly(PEGA/OA) and Poly(PEG-

SO3A/OA) coated PUs in plasma.

(43)

PDA/PEG coating Silicone PDA; PDA/PEG-OH;

PDA/PEG-COOH

Fluorescent

microscopy;

Quantitative analysis

E. coli, S. aureus and

P. aeruginosa

PDA/PEG-OH demonstrated the best antifouling

results. (44)

23

4.1.2 Polysaccharides

Due to the lack of stability of PEG coatings for long periods of time, many studies have

been made with the purpose of developing alternative polymers with antifouling properties and

longer stability. One of the alternatives are polysaccharides (35,36).

Polysaccharides are molecules with a high-molecular weight that are found in

abundance in nature. These biopolymers are constituted by more than ten repetitive units of

monosaccharides that are linked by glyosidic bonds (15,47,48). Biocompatibility, non-toxicity

and biodegradability are the main advantages presented by polysaccharides (48–50). However,

these properties are influenced by the nature of the monomers and the structure of these

biomolecules (47).

Cellulose, dextran, dermatan, heparin, chitosan and agarose are some examples of

polysaccharides (24,27,31).

4.1.2.1 Dermatan Sulfate

Dermatan sulfate (DS) is a natural glycosaminoglycan normally found in the

extracellular matrix of animal tissues, such as heart, skin and blood vessels (51–53). It has a

complex structure that is similar to that of chondroitin sulfate, however instead of glucuronic

acid it has iduronic acid (54).

This polysaccharide is negatively charged and very hydrophilic. Also, similarly to

heparin (Hep), it has anticoagulant and antithrombotic properties as well as antifouling activity

(51).

With respect to the antifouling properties of DS, there are very few studies. One of those

was developed by F. Xu et al. (51) in 2011. These authors incorporated concentrations of DS

of 0.66%, 1.5%, 3% and 10% into the side chain of PUs obtaining the copolymers PU/DS

0.66%, PU/DS 1.5%, PU/DS 3% and PU/DS 10%, respectively. The number of adherent E. coli

(BL21 strain) on PU/DS surfaces was quantified via dsDNA assay. PEG-coated PU and Agar-

coated PU were used as negative and positive controls, respectively. The obtained results

showed that the incorporation of DS into PU reduced the attachment of E. coli. Moreover,

copolymers PU/DS 0.66% and PU/DS 10% showed a density of adhered bacteria similar to the

negative control (Table 3) (51).

24

4.1.2.2 Methylcellulose

Methylcellulose (MeCe) is a natural methacrylated polysaccharide found in plants (55).

This polysaccharide is hydrophilic, possesses hydrogen bond acceptors and is neutral. So,

MeCe has good antifouling properties. (1,56). Also, it is not degradable by human enzymes

(only by cellulase, an enzyme that lacks in humans). Therefore, MeCe resists to in vivo

degradation which potentially allows long-term applications (55,57).

Additionally, this biopolymer has been used for diverse applications on biomedical field

without any safety concerns (56).

The antiadhesive properties of this polysaccharide have been investigated in different

studies. Recently, W. Mussard et al. (56) grafted a vinyl-modified MeCe on the surface of the

medical grade silicone elastomer MED-4765 by one-step reaction (Table 3). Bacterial adhesion

of four bacterial strains (E. coli MG1655, S. epidermidis ATCC 35984, P. aeruginosa ATCC

15442 and S. aureus ATCC 6538) was evaluated by SEM observation and fluorescence

microscopy. The results allowed the authors to conclude that MeCe-coated MED-4765 was able

to reduce bacterial adhesion. In fact, coated samples showed a reduction of the number of

adhered E. coli, S. epidermidis, P. aeruginosa and S. aureus of approximately 99,5%, 99%,

98.9% and 99.6%, respectively. Additionally, the MeCe grafting suppressed biofilm formation

under flow conditions (56).

Three years later, another group of researchers grafted MeCe to PDMS catheters of

totally implantable venous access ports (TIVAP) via one-step hydrosilylation in water (58). For

the in vitro and in vivo bacterial adhesion tests, the authors used the luminescent S. aureus

Xen36 and P. aeruginosa Lm1 strains and used epifluorescence microscopy and

bioluminescence activity to evaluate the adhesion of these two microorganisms (Table 3). The

obtained data from the in vitro tests demonstrated that MeCe-coated catheters significantly

reduced the adhesion of both bacteria after 24 h under static conditions and after 48 h under

continuous flow conditions. Regarding the in vivo evaluation, modified or unmodified TIVAPs

devices were implanted on male rats and maintained for 4 days. After that period of time, an

inoculum of P. aeruginosa or S. aureus was injected in the site of the implanted TIVAP, and

left there for 1 h or 3 h, respectively. The data obtained from the evaluation of the initial

attachment of bacteria demonstrated a 2-fold reduction on both S. aureus and P. aeruginosa

adhesion to the internal surface of MeCe-coated catheters. The effect of MeCe-coatings on later

stages of biofilm formation was also evaluated. For that, a 5-days monitoring of the biofilm

25

development on the internal surface of TIVAPs was made. The results showed an excellent

antibiofilm efficacy, with a 2 to 3-log reduction of bacteria colonization on modified catheters

at the end of this period (58).

4.1.2.3 Agarose

Agarose (AG) is a natural, non-ionic and hydrophilic polysaccharide that is obtained

from agar or agar-bearing seaweeds such as red algae Rhodophyta. It consists in alternating

(1→4) linked 3,6-anhydro-α-l-galactose and (1→3) linked β-d-galactose monomers (59–62).

This polysaccharide is biocompatible, non-toxic, inert and stable. Also, it presents

antifouling and non-immunogenic properties that allows its application on biomedical and

tissue engineering areas (59,60,62,63).

Aiming to investigate if AG was able to resist to bacterial adhesion, a group of

investigators coated silicone surfaces with agarose (AG) films and AG hydrogel (2% w/v) (64).

In vitro bacterial tests were performed with strains of P. mirabilis B2 (isolated from an

encrusted catheter), P. mirabilis HI4320 (wild-type) and P. mirabilis HI4320 ure- (a urease-

negative mutant). The attached bacterial cells were observed in a parallel-plate flow-cell

chamber and quantified in terms of the number of cells per high power field and per cm2 of

surface (Table 3). Bacterial tests were performed with urine or phosphate buffer. The

experimental results obtained from urine cultures using P. mirabilis HI4320 ure- revealed a

reduction on bacterial adhesion of ≈ 94%. After 6 h, the bacterial adhesion in both media was

significantly lower compared to bare silicone. Relatively to AG hydrogel coating, data from

tests with buffer revealed a low adhesion of P. mirabilis B2, and in urine cultures no bacterial

adhesion was observed for the first 4 h. In spite of the good results demonstrated by both AG

films and hydrogels, AG hydrogel demonstrated the best antifouling properties (64).

4.1.2.4 Chitosan

Chitosan (CH) is a natural polysaccharide that is obtained by partial deacetylation of

chitin (1,30,54,65).

This biopolymer is composed by units of N-acetyl-D-glucosamine and D-glucosamine

linked via β-(1,4) glycosidic bonds. The amine groups in its structure confers a positive charge

26

to CH that interacts with negatively charged bacteria, disrupting the cell membrane, being

through this mechanism that CH reveals bactericidal activity (49,54,66).

CH has many applications in the biomedical field once it is biocompatible,

biodegradable, non-toxic and low-immunogenic (49,54,67).

Several studies have been performed aiming to characterize the bactericidal activity of

CH through the last years. However, antiadhesive properties of this polysaccharide are poorly

investigated. Nevertheless, in 2012, R. Wang et al. (68) performed a study with the purpose of

evaluating the ability of CH in inhibiting E. coli and P. mirabilis adhesion and biofilm

formation on medical grade silicone surfaces (Table 3). Silicone samples were coated with PDA

and then grafted with carboxymethyl chitosan (CMCS) through Michael addition and Schiff-

base reactions. The obtained polymer was named as PDA-CMCS. After this step, some samples

suffered a crosslink of the CMCS layer, obtaining PDA-CMCS-X polymers. Bacterial tests

were performed under static and flow conditions, and the used microorganisms were E. coli

(ATCC DH5α) and a strain of P. mirabilis (ATCC 51286) isolated from a patient with catheter-

associated urinary tract infection (CAUTI). The results obtained from SEM and fluorescence

microscopy allowed to conclude that PDA-CMCS and PDA-CMCS-X coatings were able to

reduce P. mirabilis adhesion in 88% and 90%, respectively, and E. coli adhesion in ≈ 90% on

both surfaces. Additionally, under static conditions the authors observed biofilm disruption on

both PDA-CMCS-X and PDA-CMCS surfaces. Under flow conditions, PDA-CMCS surface

revealed ≈ 20% coverage by P. mirabilis and ≈ 13% by E. coli biofilms (68).

Although the statement on antifouling properties by the authors, it cannot be forgotten

that CH is well known by its antimicrobial characteristics. So, part of the antiadhesive results

verified by the authors may be due to the interaction of this polysaccharide with the membrane

of bacteria, with consequent disruption and death, resulting in a reduction of the number of

viable bacteria that are capable of adhere to the surfaces.

4.1.2.5 Heparin

Hep is a natural and anionic polysaccharide of animal origin. The negative charge of

this biopolymer is due to the presence of carboxylate and sulfate groups in its structure

(48,53,54).

Hep is well known by its good anticoagulant and antithrombotic activities, since it is

able to inhibit both intrinsic and extrinsic clotting cascades (53,69,70). Such properties

27

increased the interest by using this polysaccharide on biomedical applications, such as coating

blood-contacting biomedical devices (48,71).

Different techniques to immobilize this polysaccharide to these surfaces includes

covalent attachment and electrostatic self-assembly (72).

In addition to the properties already mentioned above, it was proved that Hep also

presents the ability of inhibiting bacterial adhesion and biofilm formation (27,54,69,71,73).

This inhibition occurs because Hep negative charge repels negative charged bacteria through

repulsive forces (27,69,71). Furthermore, some Gram-positive bacteria have adhesins that are

able to recognize fibronectin (the main adsorbed protein on the surface of blood-contacting

devices). Thus, Hep coatings leads to a reduction on fibronectin adsorption with consequent

prevention of bacterial attachment to the surfaces (73).

Focusing on the resistance to bacterial adhesion of this polysaccharide, Hep was

adsorbed to the surface of Bionate®-PIME samples (74). Bionate®-PIME is a polycarbonate-

urethane (PCU) with low amounts of diamino-diamide-diol (PIME) in the main backbone.

PIME promotes the binding of Hep to PCU surfaces mainly via ionic bonds, which results in a

major stability of Hep grafting. Strains of S. aureus (Cowan 1 and 8325-4) and S. epidermidis

(RP62A and HB) were used on the in vitro bacterial adhesion assays. The results were assessed

by monitorization of the absorbance at 595 nm through a 3-(4,5) dimethylthiazol-2-yl-2,5

diphenyl tetrazolium bromide (MTT) test and by SEM observation. Two medical-grade PCUs

were used as controls: Bionate® and Carbothane®. The obtained data revealed that heparinized

Bionate®-PIME surfaces reduced S. epidermidis and S. aureus colonization. In fact, SEM

images of Hep-treated surfaces demonstrated small size and dispersed clusters of bacteria,

compared to the untreated surfaces that showed a dense and uniform layer of bacteria (74).

Recently, some researchers have also developed studies that associate Hep with other

polysaccharides to coat the surface of some biomaterials with the aim of evaluating the

antifouling properties of these associations.

One of those studies was performed in 2014 by M. Li et al. (24) that covalently

immobilized and crosslinked an acrylated AG layer on both silicone films and PD silicone

catheter surfaces (Table 3). Some of the samples additionally suffered an incorporation of

methacrylated Hep into the crosslinked AG. For this grafting, silicone surfaces were previously

activated by O2 plasma or O3 treatment and then the acrylated AG and methacrylated Hep

suffered an ultraviolet (UV) or heat-induced immobilization and crosslinking. The obtained

28

surfaces were named as silicone-g-AG and silicone-g-AG-Hep. Bacterial adhesion tests were

performed with S. aureus (ATCC 25923), E. coli (ATCC DH5α) and P. aeruginosa (PAO1) as

model bacteria, and the results were assessed by fluorescence microscopy and SEM

observation. The authors concluded that both AG and AG-Hep-modified films significantly

reduced S. aureus, E. coli and P. aeruginosa adhesion by ≈ 2.4, ≈ 3.3 and > 2 folds, respectively.

E. coli and S. aureus biofilm formation was prevented in both modified surfaces. Also, with the

increase of UV-induced grafting time (120 minutes), E. coli and S. aureus adhesion reduced to

≈ 99% and ≈ 98%, respectively. Similar to modified silicone films, AG and AG-Hep modified

catheters showed a reduction on E. coli adhesion of more than 3 orders of magnitude (24).

In another study, silicone films were modified with mesoporous silica nanoparticles

(MSNs) loaded with AG or AG and Hep via electrostatic interaction (75). The obtained

nanoparticles were named as Agarose-Loaded Mesoporous Silica Nanoparticles (AMSNs) and

Agarose and Heparin-Loaded Mesoporous Silica Nanoparticles (AHMSNs). For the in vitro

assays, samples of bare silicone, AMSNs-Si and AHMSNs-Si were cultured with E. coli and S.

aureus. The bacterial adhesion was evaluated by SEM observation (Table 3). It was concluded

that both AMSN and AHMSN coatings significantly reduced microbial adhesion on silicone

surfaces. Also, besides the resistance to bacterial adhesion, AHMSN coating also demonstrated

good hemocompatibility due to the presence of Hep (75). This last property represents an

advantage relatively to the AMSNs-coatings.

Additionally, a combination of Hep with CH was also studied by F. Kara et al. (69) to

evaluate the antibacterial and antiadhesive properties of this association (Table 3). For this, the

authors synthetized new hexamethylene diisocyanate based PUs (PUh) by using aliphatic

hexamethylene diisocyanate (HDI) and polyol. Then, PUs surfaces were plasma activated and

CH and Hep were covalently immobilized onto these surfaces via a stepwise process. The

investigators used concentrations of 5 mg/mL and 20 mg/mL of CH, and a concentration of 5

mg/mL of Hep, obtaining the polymers PUh-CH-0.5, PUh-CH-2.0 and PUh-CH-Hep,

respectively. Regarding bacterial adhesion tests, S. aureus (ATCC 25923), S. epidermidis

(ATCC 12228), E. coli (ATCC 11229) and P. aeruginosa (ATCC 27853) were used as bacteria

models. The adhered bacteria were observed by SEM and quantified by counting the number

of CFUs/cm2. The authors concluded that both PUh-CH-0.5 and PUh-CH-2.0 surfaces were

able to reduce the adhesion of the four types of tested bacteria after 4 h-incubation. Also,

comparing with bare PUh, PUh-CH-Hep demonstrated the best antifouling results, showing a

29

reduction of 97%, 90%, 98% and 99% on E. coli, P. aeruginosa, S. aureus and S. epidermidis

adhesion, respectively (69).

30

Table 3 – Use of polysaccharides as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test the

strategy Tested microorganisms Results Ref.

Ty

pe

of

cath

eter

Uri

na

ry MeCe grafting PDMS MED-4765-MeCe

Fluorescence microscopy;

SEM

E. coli MG1655, S.

epidermidis (ATCC 35984),

P. aeruginosa (ATCC 15442)

and S. aureus (ATCC 6538)

≈ 99% reduction on bacterial adhesion.

Suppression of biofilm formation. (56)

CMCS coating Silicone PDA-CMCS;

PDA-CMCS-X

SEM; Fluorescence

microscopy; Number of

CFUs/cm2

E. coli (ATCC DH5α) and P.

mirabilis (ATCC 51286)

≈ 90% reduction of E. coli and P. mirabilis

adhesion. Disruption of biofilms. (68)

Va

scu

lar

DS PU

PU/DS 0.66%; PU/DS

1.5%; PU/DS 3.0%;

PU/DS 10%

dsDNA assay for adherent

bacteria E. coli (BL21 strain)

< 300 ng DNA/sample with DS contents of 0.66%

and 10% (versus ≈ 700 ng DNA/sample in control) (51)

MeCe grafting PDMS Si-MeCe

Epifluorescence

microscopy;

Bioluminescence activity;

Number of CFUs/cm2

S. aureus Xen36, P.

aeruginosa Lm1

2-fold reduction on bacterial adhesion. 2 to 3-log

reduction of biofilm formation after 5 days. (58)

CH and Hep

immobilization PU

PU-CH-0.5; PU-CH-

2.0; PU-CH-Hep

Number of CFUs/cm2;

SEM

S. aureus (ATCC 25923), S.

epidermidis (ATCC 12228),

E. coli (ATCC 11229) and P.

aeruginosa (ATCC 27853)

90% to 99% reduction of bacterial adhesion in

PUh-CH-Hep. (69)

Heparinizable

modified PCU PCU Bionate®-PIME-Hep MTT test; SEM

S. aureus Cowan 1 and 8325-

4, S. epidermidis RP62A and

HB

2 to 4-log reduction of S. aureus and S.

epidermidis adhesion on Bionate®-PIME-Hep. (74)

PD

Acrylated AG

and/or

Methacrylated Hep

coating

Silicone Si-g-AG;

Si-g-AG-HEP

SEM; Fluorescence

Microscopy; Number of

CFU/cm2

S. aureus (ATCC 25923), E.

coli (ATCC DH5α) and P.

aeruginosa (PAO1)

Reduction of bacterial adhesion > 2 orders of

magnitude and ≈ 98% after 2 h of UV radiation. (24)

AG coating Silicone

Si-AG;

Si-AG hydrogel (2%

w/v)

Parallel-plate flow-cell

chamber; Number of cells

per high power field and

per cm2 of surface

P. mirabilis B2, P. mirabilis

HI4320 (wild-type) and P.

mirabilis HI4320 ure-

≈ 94% and > 80% reduction of P. mirabilis

HI4320 ure- and P. mirabilis B2 adhesion,

respectively.

(64)

MSNs coating with

AG and/or Hep Silicone

AMSNs-Si;

AHMSNs-Si SEM E. coli and S. aureus

Both tested MSNs significantly reduced S. aureus

and E. coli adhesion. (75)

31

4.1.3 Polyacrylamide and polyacrylates

Polyacrylamide (PAAm) is a neutral and polar macromolecular polymer that presents

good stability, biocompatibility, hydrophilicity and at the same time is neither toxic nor

immunogenic (66,76–80).

Although there are few studies that used PAAm hydrogels or brushes to coat the surface

of biomaterials such as silicon, it has been proved that this polymer is able to resist to bacterial

and protein adhesion (35,66,76,78,81).

With the aim of evaluating the antifouling properties of this polymer, I. Fundeanu et al.

(82) covalently attached amino-poly(o-amino-p-xylylene-co-p-xylylene) (amino-PPX)-PAAm

brushes to silicone rubber samples by atom transfer radical polymerization (ATRP) (Table 4).

For the performance of bacterial adhesion tests two bacterial strains were selected, S. aureus

(ATCC 12600) and a fluorescent strain of E. coli (E. coli 3.14) and the evaluation was

conducted in a parallel plate flow chamber under moderate flow conditions. Fluorescence

microscopy and initial deposition rates were used to evaluate the strategy. The obtained results

showed a reduction of S. aureus and E. coli adhesion of 93% and 99%, respectively, on amino-

PPX-PAAm brush-coated samples when compared to bare silicone rubber. Regarding the

adhesion kinetics, S. aureus attached more than 10-fold slower after the amino-PPX–PAAm

brush-coating (207 ± 58 cm-2 s-1 versus the initial deposition rate of 2515 ± 812 cm-2 s-1), and

E. coli adhesion was not detectable during the 4 h of the experiment (versus an initial deposition

rate of 34 ± 332 cm-2 s-1) (82).

In another study, the resistance to bacterial adhesion of some polyacrylates/acrylamides

was investigated (83). For that a polymer microarray technique was used to identify polymers

with resistance to adhesion of some clinically relevant bacteria. Of a library of 381

polyacrylates/acrylamides and PUs, only two polymers were chosen: poly(methylmethacrylate-

co-dimethylacrylamide) (PA13) and poly(methoxyethylmethacrylate-co-

diethylaminoethylacrylate-co-methylmethacrylate) (PA515). Polymer

poly(hydroxyethylmethacrylate-co-dimethylaminoethylmethacrylate) (PA155) was used as

positive control. The authors coated pieces of a PU and silicone vascular catheters (Cath-1 and

Cath-2, respectively) with PA13, PA515 and PA155. The efficacy of the coatings, was

evaluated against two mixtures of bacteria: BacMix-1 (i.e. K. pneumoniae, Staphylococcus

saprophyticus and S. aureus) and BacMix-2 (i.e. K. pneumoniae, Streptococcus mutans, S.

aureus and Enterococcus faecalis). Except for S. mutans (NCTC 10923) strain, the other

32

bacteria were isolated from infected medical devices. The results from bacterial tests were

evaluated by confocal microscopy analysis and by SEM observation after 72 h and 12 days

incubation, respectively. The obtained results showed that coating Cath-1 surfaces with PA515

reduced the attachment of bacteria in about 64% for BacMix-2, and PA13 coating showed an

≥ 96% reduction of bacterial adhesion for both BacMix-1 and BacMix-2. Which concerns to

Cath-2, P515 coating has displayed a 19% reduction of bacterial adhesion with BacMix-1, and

at least 82% reduction of bacterial adhesion with both bacterial mixtures on PA13-coated

pieces. It was also concluded that PA13 and P515 had good potential as antiadhesive coatings

for medical devices. However, PA13 was the polymer that presented better antiadhesive

efficacy, making of it the best choice for catheter coating (Table 4) (83).

33

Table 4 – Use of PAAm and polyacrylates as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Va

scu

lar

Amino-PPX-PAAm

brushes

Silicone

rubber

Amino-PPX-PAAm

silicone rubber

Fluorescence

microscopy;

Adhesion kinetics

S. aureus (ATCC

12600) and E. coli

3.14

Reduction of E. coli and S. aureus adhesion in 99%

and 93%, respectively. (82)

Polyacrylates/

acrylamides

coatings

Polyurethane

and Silicone PA115; PA13; PA515

Confocal microscopy

analysis; SEM

K. pneumoniae, S.

saprophyticus, S.

aureus, E. faecalis,

and S. mutans

(NCTC 10923)

Observed a reduction between 19% and 64% with

PA515 and between 82% and 96% with PA13. (83)

34

4.2 Zwitterionic Polymers

Zwitterions are neutral molecules that have both negatively and positively charged

groups in the same molecular unit (18,40,46). They are very hydrophilic since they attach to

water molecules through ionic bonds (1,15,31,33).

These polymers are biocompatible, stable against oxidation and, as several studies have

demonstrated, they are also resistant to bacterial and nonspecific proteins adhesion. Such

properties make of zwitterions good options to antifouling applications on biomedical field

(1,32,46,70,84–89).

Zwitterions may be grouped into betaines and polyampholytes. However, the most

commonly studied zwitterions are betaines, which are examples sulfobetaine (SB),

carboxybetaine (CB) and phosphorylcholine (PC) (31,36,90–92).

4.2.1 Betaines

Of betaines, SB is the one that presents more studies with potential application on

catheters or catheter-materials, followed by PC.

One of those studies was performed by R. Smith et al. (93) in 2012. This group of

researchers grafted a non-leaching polymeric sulfobetaine (poly-SB) on the internal and

external surfaces of a vascular catheter by redox polymerization process. To evaluate the

antifouling properties of poly-SB grafting to PU catheters, the authors used suspensions of E.

coli (ATCC 700928) and S. aureus (ATCC 25923) to contaminate samples of bare and poly-

SB-coated catheters (Table 5). After a period of 24 h, the number of CFUs/cm2 on both internal

and external surfaces of the samples was determined. The obtained data demonstrated that poly-

SB coating reduced bacterial adhesion on 97% to 99.9% on the external surface of poly-SB-

coated catheters. Relatively to the internal surface of catheters, this coating reduced E. coli and

S. aureus adhesion on 96% and 97%, respectively. The authors also examined bacterial

attachment under in vivo conditions. For this purpose, uncoated and poly-SB-coated samples

were first exposed to S. aureus cultures for 2 h and then were implanted into small subcutaneous

pockets in healthy rabbits. Rabbits were maintained for 3 to 5 days and then euthanized. The in

vivo test results allowed to conclude that poly-SB coated catheters reduced S. aureus adhesion

on 97% compared to uncoated sample (93).

35

In another study, PDMS urinary catheters surfaces were modified with sulfobetaine

methacrylate (SBMA) by radical polymerization (19). The antiadhesive and antibiofilm

efficacy of SBMA-coating was evaluated against two bacteria models, P. aeruginosa (ATCC

10145) and S. aureus (ATCC 25923), under static and dynamic conditions. The obtained results

from static condition tests were assessed by bacterial viability assessment and fluorescence

microscopy. Compared to bare PDMS catheters, SBMA-coated catheters showed a S. aureus

and P. aeruginosa adhesion of 42% and 35%, respectively. It was also performed a dynamic

test by inserting both bare and SBMA-coated catheters into an artificial bladder model filled

with artificial urine contaminated with the two types of bacteria. After 7 days, it was quantified

the number of adhered bacteria on three different parts of the catheters: tip, balloon and urethra

(the last one is referred to the portion below the balloon area). In case of P. aeruginosa, urethra

of SBMA-coated catheters revealed a biofilm reduction of ≈ 80% compared to bare catheters.

Also, it was registered a reduction of ≈ 90% in S. aureus biofilm formation in balloon portion

of the SBMA-coated samples (Table 5) (19).

The efficacy of SB silane (SBSi) on resisting against bacterial adhesion was also

evaluated by S. Yeh et al. (94). In that way, the researchers modified the surface of PDMS

samples by covalent silanization of SBSi (Table 5). For the bacterial adhesion tests two bacterial

strains were used (i.e. S. epidermidis and P. aeruginosa). The results were assessed by

fluorescent microscopy. The authors concluded that the modification of PDMS surfaces with

SBSi significantly reduced adhesion of both bacteria. In fact, comparing to bare PDMS, the

coated samples prepared on day 0 (PDMS-SBSi-0D) presented a reduction on S. epidermidis

and P. aeruginosa adhesion of 99.78% and 99.86%, respectively. In the aged samples (PDMS-

SBSi-30D), P. aeruginosa adhesion remained almost the same as the fresh samples. However,

the resistance to S. epidermidis adhesion registered a slight decrease. Nevertheless, the general

antifouling rates of SBSi-coated samples were over 90% compared to bare PDMS (94).

More recently in 2017, C. Xing et al. (44) synthetized copolymers bearing PC and active

ester groups (PMENs) by using monomers of 2-methacryloyloxyethyl phosphorylcholine

(MPC) and p-nitrophenoxycarbonyloxyethyl methacrylate (NPCEMA) (Table 5) according to

their reported method in 2012 (95). The synthetized polymer was named of PDA/PMEN10.

Aiming to evaluate the efficacy of PDA/PMEN10 on preventing bacterial adhesion, the authors

immobilized this zwitterionic polymer on the surface of PDA-precoated silicon wafers and used

E. coli, S. aureus and P. aeruginosa suspensions to perform bacterial tests. Fluorescence

microscopy images allowed the authors to conclude that PDA/PMEN10-coated surfaces

36

reduced the adhesion of the three types of tested bacteria in 99.8%, compared with the bare

silicon wafers (44).

Despite the good antifouling properties demonstrated by betaines, these compounds are

prone to hydrolysis under physiological conditions, since they have ester or amide bonds in

their structures (86,96).

With the purpose to find an alternative to this problem, Y. Sun et al. (86) developed a

more stable and non-degradable alternative (Table 5). For this, this group of investigators first

modified a silicon wafer with triethoxyvinylsilane obtaining reactive double bonds on the

surface. Then, polymer 4-vinyl pyridine (P4VP) was grafted onto the silicon surface by

“grafting from” technique followed by 1,3-propanesultone quaternization, obtaining the

substrate Si-P4VP-psl. Aiming to evaluate the resistance to bacterial adhesion of this

zwitterionic coating, the authors used an E. coli (ATCC 25922) suspension to perform bacterial

tests. SEM images revealed that Si-P4VP-psl surface presented less adhered bacteria than bare

silicon wafer. However, the authors pointed out that the long term effectiveness of this brushes

must be explored in the future (86).

37

Table 5 – Use of zwitterions as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Uri

na

ry

SBMA coating PDMS SBMA

Fluorescence

microscopy; Bacterial

viability test

P. aeruginosa

(ATCC 10145)

S. aureus (ATCC

25923)

58% to 65% reduction of bacterial adhesion.

Reduction of biofilm formation in ≈ 80% for P.

aeruginosa and ≈ 90% for S. aureus.

(19)

Zwitterionic P4VP-

psl brush Silicone Si-P4VP-psl SEM

E. coli (ATCC

25922) Low E. coli adhesion was observed. (86)

SBSi PDMS SBSi Fluorescence

microscopy

S. epidermidis and

P. aeruginosa

Reduction on S. epidermidis and P. aeruginosa

adhesion of 99.78% and 99.86%, respectively, at

day 0. Antiadhesion rates over 90% after 30 days.

(94)

Va

scu

lar

PDA/PMEN10

coating Silicone PDA; PDA/PMEN10

Fluorescent

microscopy;

Quantitative analysis

E. coli, S. aureus

and P. aeruginosa

PDA/PMEN10 reduced bacterial adhesion and

proliferation in 99.8%. (44)

Poly-SB Polyurethane Poly-SB Number of CFUs/cm2

E. coli (ATCC

700928) and S.

aureus (ATCC

25923)

97% to 99.9% and 96% to 97% reduction of

microorganisms’ adhesion on the internal and

external surfaces, respectively.

(93)

38

4.3 Other antifouling coatings

4.3.1 Amphiphilic polymers

Amphiphilic (also known as amphipathic) polymers are molecules with high molecular

weight that have a hydrophilic and a hydrophobic portion on its structure (85,97,98).

These polymers can provide a diversity of properties such as flexibility, durability,

higher stability and resistance to biofouling (85,91,99,100). These characteristics increased the

interest on the application of these molecules on diverse areas such as biomaterials and medical

care (97).

The coating of surfaces with amphiphilic polymers may be achieved through grafting

process or blending with bulk materials, for example (98,99).

Some studies have been recently developed with the purpose of investigating the

antifouling properties of these type of polymers. One of those studies was performed by J. Jiang

et. al (85) that developed a novel amphiphilic PDMS-based PU networks tethered with CB

(Table 6). These networks were synthetized by a crosslink reaction of di/tri isocyanate and t-

butyl protected CB (PCB) as the hard domain, and siloxane diol as the soft domain. For the

synthesis of PU networks, the authors used PDMS:PCB molar ratios of (4.5:0), (3:1.5), (1.2:3.3)

and (0:4.5), obtaining the networks PU-0, PU-1, PU-2 and PU-3, respectively. Finally, the

samples suffered an acid hydrolysis so the t-butyl group could be removed and the zwitterionic

properties of CB were acquired by the samples. For the bacterial adhesion tests, an E. coli

suspension was used, and the density of bacterial cells was assessed by fluorescence microscopy

images. The obtained results allowed to conclude that all the synthetized amphiphilic PU

networks demonstrated excellent results on resisting to E. coli adhesion, compared to epoxy

resin (control). Comparing with control ((26.6 ± 0.8) x 105 cells/cm2), PU-3 was the sample

with the best antifouling properties, with an adherent bacteria density of (1.8 ± 0.4) x 105

cells/cm2, followed by PU-2 and PU-0 that showed an adherent bacteria density of (3.2 ± 0.3)

x 105 cells/cm2 and (11.5 ± 0.5) x 105 cells/cm2, respectively (85).

On the same year, H. Keum et al. (28) used a long alkyl chain dodecyl methacrylate

(DMA), a short poly(ethylene glycol) methacrylate (PEGMA), and an acrylic acid (AA) on the

molar ratio of (3.5:3.5:3) to synthetize the amphiphilic polymer poly(DMA-mPEGMA-AA) by

radical polymerization (Table 6). Then, the authors coated PDMS samples with concentrations

of poly(DMA-mPEGMA-AA) of 1, 2, 5, 10 or 20 mg/mL. To evaluate the antifouling efficacy

39

of coated PDMS samples, two microbial strains, S. aureus (ATCC 25923) and a fluorescent

mCherry-marked E. coli, were used to incubate the samples for 8 h and 24 h. WST-1 assay

results demonstrated that independently of poly(DMA-mPEGMA-AA) concentration, all the

tested surfaces showed a reduction on S. aureus adhesion (only 1 to 6% adhesion compared to

the uncoated PDMS surfaces). Relatively to mCherry-marked E. coli, fluorescence microscopy

demonstrated a reduced bacterial adhesion on all coated surfaces. The researchers also

evaluated the long-term efficacy of this coating for a period of 7 days. SEM images revealed

few bacterial adhesion and colonization on the surface of coated PDMS. Tests conducted on

silicone urinary catheters under flow conditions revealed that poly(DMA-mPEGMA-AA)

coating was able to reduce S. aureus adhesion in approximately 77% at the end of 2 days. In

vivo assays experiments were also performed. For this, the authors implanted uncoated or

polymer-coated catheters subcutaneously at the right flank of mice and 2 days later they injected

phosphate buffered saline (PBS) or S. aureus suspension into the site containing the catheters.

After 4 days, all the poly(DMA-mPEGMA-AA)-coated catheters previously inoculated with S.

aureus revealed low microbial colonization, with a optical density (O.D.) of 0.102 (versus 0.770

of the uncoated samples) (28).

4.3.2 Modified Polyurethanes

Segmented PUs are constituted by alternating hard and soft domains. The hard domains

are often formed by diisocyanates and chain extenders, and the soft domains are formed by

macrodiols (27,101–105).

These PUs have good blood-compatibility, present good flexibility and mechanical

strength conferred by the soft and hard domains, respectively (27,73,103,106). Such properties

have increased the use of these polymers on the manufacture of medical devices (69,73,106).

To date, very few researchers have modified these polymers to create PUs able to reduce

microbial adhesion.

Still, few studies were developed and one of those was in 2012 by Francolini et al. (73)

who synthetized new heparin-mimetic segmented PUs, by adding sulfate or sulfamate groups

(known to be responsible for the Hep activity) to the side chain of carboxylated PUs (PEUA)

(Table 7). The first step in this synthesis was the amidation of PEUA with ethanolamine (EA),

serinol (S) or ethylene diamine (ED), obtaining the polymers PEUEA, PEUS and PEUED,

respectively. In the second step, PEUEA and PEUS reacted with pyridine-SO3 adduct and

40

PEUED reacted with DMF-SO3, resulting in the sulfated polymers PEUEA–SO3H, PEUS–

SO3H and PEUED–SO3H, respectively. A suspension of S. epidermidis (ATCC 35984) was

used to perform in vitro bacterial tests. The obtained data from SEM observation demonstrated

that sulfonated polymers completely inhibited bacterial colonization, and PEUS-SO3H (with a

higher composition in –SO3H groups) demonstrated excellent antifouling properties (73).

The effect of polymer hydrophilicity and the degree of hard/soft domain separation on

antifouling properties of PUs was also assessed. For this, Francolini et al. (106) synthetized

four new segmented PUs with the same hard domain, but a variable soft domain (Table 7). The

hard domain was constituted by methylene-bis-phenyl-isocyanate (MDI) and

dihydroxymethyl-propionic acid (DHMPA). On the other hand, the soft domain was constituted

by one of the following three macrodiols: polycaprolactide (PCL), poly-l-lactide (PLA) or

polypropylenoxide (PPO). For the PCL and PLA-based PUs the soft domain content per repeat

unit of polymer was 0.50 (expressed as molar fraction) (PCL0.50-UA and PLA0.50-UA,

respectively). With the aim of evaluating the influence of soft domain content in the polymers,

PPO-based PUs were synthetized with two different contents of soft domain: 0.50 (PPO0.50-

UA) and 0.65 (PPO0.65-UA). The antifouling properties of segmented PUs was studied using a

suspension of S. epidermidis (ATCC 35984). The results obtained by SEM imaging allowed

the authors to conclude that the type of soft domain had influence on bacterial adhesion, with

PLA0.50-UA showing the best antifouling results (2 x 102 CFUs/cm2), followed by PCL0.50-UA

(1 x 105 CFUs/cm2) and PPO0.50-UA (1 x 107 CFUs/cm2). Relatively to the amount of soft

domain, PPO0.65-UA demonstrated only 1-log reduction on S. epidermidis adhesion comparing

to PPO0.50-UA. Additionally, after 24 h incubation, it was observed the formation of a mature

S. epidermidis biofilm on PPO0.50-UA surface, only a few single cells or small bacterial

aggregates on PCL0.50-UA and no S. epidermidis colonization on PLA0.50-UA surface, making

of this last polymer the best antifouling PU of the four tested polymers (106).

4.3.3 Slippery Liquid-Infused Porous Surfaces

Slippery Liquid-Infused Porous Surfaces (SLIPS) are super-hydrophobic surfaces that

were inspired on Nepenthes pitcher plant. This plant use a layer of liquid water at its surface,

creating a “slippery” surface that prevents the attachment of insects (35,107,108).

This technology includes a micro/nanopatterned surface that is infiltrated with a

lubricating liquid (normally liquid perfluorocarbons) that confers antiadhesive properties to the

41

surface (29,92,107,108). The lubricating liquid has to be stable, non-toxic and with high affinity

to the substrate (Figure 3) (29,35,100).

SLIPS surfaces started to be studied in non-medical field. More recently, the interest in

the possible application of this strategy on biomedical devices increased the number of studies

in this area (15,35,92,108).

One of those studies was performed by A. Epstein et al. (100) that infused a porous

Teflon® membrane with Krytox® 103, a perfluorinated liquid that acts as lubricating fluid

(Table 8). P. aeruginosa PA14, S. aureus SC01 and E. coli ZK2686 strains were used to

perform bacterial tests under static and flow conditions. The results were assessed by

fluorescence microscopy and crystal violet absorbance. The results showed very low microbial

adhesion on SLIPS surfaces. Under static conditions, SLIPS surfaces demonstrated a reduction

of S. aureus and E. coli adhesion of 97.2% and 96%, respectively. After 7 days under flow

conditions, SLIPS surfaces reduced P. aeruginosa biofilm formation in about 99.6%, and the

fluorescence signal reduced ≈ 98% (100).

Two years later, another group of researchers covalently linked liquid perfluorocarbon

(TLP) on the surface of PVC medical tubing and polyethylene terephthalate (PET) samples,

and then infused it with a mobile layer of perfluorodecalin (107) (Table 8). To determine the

antifouling efficacy of these SLIPS surfaces, P. aeruginosa and E. coli (ATCC 8739) were used

as bacteria models on bacterial adhesion tests. The results were assessed by SEM and crystal

violet staining, and it demonstrated that TLP-coated PET significantly reduced P. aeruginosa

and E. coli adhesion. Also, after 6.5 weeks of incubation, TLP-coated PVC tubes showed an 8-

fold reduction on P. aeruginosa biofilm development (107).

Nano/micropatterned surface Lubricating

Liquid

Figure 3 – Representation of a SLIPS surface

42

4.3.4 Sharklet topography

Sharklet technology is an antifouling strategy inspired on the surface topography of

sharkskin (15,35,38,109,110). In fact, the skin of this aquatic animal presents a diamond-like

micro-geometry that prevents the adhesion of algae and bacteria (35,90,111).

The application of this technology on biomedical surfaces has been studied through the

years with the aim of reducing bacterial adhesion to these devices (15,35,110).

In one of these studies, K. Chung et al. (109) developed a Sharklet AF™ topography

with protruding features on the surface of PDMS samples, by replication of silicon wafer molds.

The used design presented 2 µm feature width and spacing, and 3 µm height. Aiming to evaluate

the antiadhesive and antibiofilm properties of this strategy, Sharklet AF™ and bare PDMS

samples were incubated in a S. aureus (ATCC 29213) suspension (Table 9). The results were

assessed by SEM at the end of 0, 2, 7, 14 and 21 days. The obtained data showed a bacterial

coverage on Sharklet AF™-PDMS of 0% on day 0, 7% on day 14 and 35% on day 21.

Additionally, only at day 21 S. aureus biofilms started to appear in small and isolated areas of

Sharklet AF™ surfaces. Until that day no evidence of early-stage biofilm formation was

observed (109).

Aiming to evaluate the ability of Sharklet micropattern surfaces on prevention of E. coli

(ATCC #700336) adhesion and biofilm formation on silicone urinary catheters, S. Reddy et al.

(112) developed three different Sharklet topographies on the surface of silicone samples: a

positive Sharklet with protruding features (2 µm width and spacing), an inverse Sharklet with

recessed features (2 µm width and spacing) and a Sharklet with protruding features (10 µm

width and 2 µm spacing). The bacterial adhesion and biofilm formation after 24 h, 48 h, 3 days

and 7 days was assessed by SEM. The results allowed the authors to conclude that all the tested

Sharklet surface modifications significantly reduced E. coli adhesion and area coverage. Also,

the colonies size reduced ≈ 76% on Sharklet surfaces with 2 x 2 and 10 x 2 features and 80%

on inverse Sharklet when compared to unmodified silicone (Table 9) (112).

Another group of investigators developed two types of Sharklet micropatterns on the

surface of thermoplastic PUs samples by thermal embossing (111). The tested topographies

were Sharklet micropatterns with recessed or protruding features (3 µm tall or deep and 2 µm

width and spacing). The obtained samples were named as -3SK2×2 and +3SK2×2, respectively.

In vitro bacterial tests were performed with S. aureus (ATCC 6538) and S. epidermidis (ATCC

35984) strains, and the obtained data were assessed by fluorescence microscopy and confocal

43

laser scanning microscopy (LSM) (Table 9). The obtained results demonstrated that, after a

period of 18 h-incubation, -3SK2x2 topography reduced S. aureus adhesion in 63% to 70%,

and S. epidermidis attachment in about 71%. Besides, both +3SK2×2 and -3SK2×2 PU surfaces

revealed smaller bacterial colonies than control PU (111).

More recently, in 2017, A. Magyar et al. (113) and V. Arthanareeswaran et al. (114)

published the results from a phase I randomized clinical trial that had the aim of investigate the

efficacy of Sharklet catheters on prevention of CAUTIs. In this study, fifty eligible adult men

who needed urethral catheterization during a period of 3 to 30 days were randomized into 2

groups. The first group was catheterized with unmodified silicone Foley catheters, and the

second group with Sharklet micropattern catheters. After catheters removal, bacterial adhesion

and biofilm formation on three different parts of the catheters (tip, middle and base) were

evaluated by SEM imaging. The incidence of symptomatic urinary tract infection (UTI), pain,

discomfort and the presence of bacteria in urine were also investigated. Which concerns to

Sharklet micropattern catheters, SEM images revealed a significant reduction on biofilm

formation. Also, the pain and discomfort in patients using the topographically modified

catheters were seriously lower when compared with patients using unmodified catheters. These

results have revealed promising and may lead to more studies in the area of topographical

modifications of catheter surfaces in the future (113,114).

44

Table 6 – Use of amphiphilic polymers as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Uri

na

ry

Poly(DMA-

mPEGMA-AA) PDMS

Poly(DMA-mPEGMA-

AA)-

Coated PDMS

WST-1 assay; SEM;

Fluorescence

microscopy; Gram

staining; Optical

Density (O.D.) values

S. aureus (ATCC

25923) and

mCherry-tagged E.

coli

1 to 6% bacterial adhesion. Reduction on S. aureus

adhesion in ≈ 77% under flow conditions. (28)

Va

scu

lar

Amphiphilic PDMS-

based PU film

tethered with CB

Polyurethane PU-0; PU-1; PU-2; PU-3 Fluorescence

Microscopy E. coli

Number of Adhered cells/cm2 of: (11.5 ± 0.5) x 105

on PU-0, (3.2 ± 0.3) x 105 on PU-2 and (1.8 ± 0.4) x

105 on PU-3 (versus (26.6 ± 0.8) x 105 of control).

(85)

Table 7 – Use of modified PUs as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Va

scu

lar

Heparin-mimetic

polyurethanes Polyurethane

PEUA; PEUEA; PEUS;

PEUED; PEUEA-SO3H;

PEUS-SO3H; PEUED-

SO3H

Number of

CFUs/cm2; SEM

S. epidermidis

(ATCC 35984)

≈ 3-fold reduction of bacterial adhesion on -SO3H

group-containing polymers relatively to PEUS. (73)

Segmented

polyurethanes Polyurethane

PCL0.50-UA

PLA0.50-UA

PPO0.50-UA

PPO0.65-UA

Number of

CFUs/cm2; SEM

S. epidermidis

(ATCC 35984)

Antifouling efficacy: PLA0.50-UA (102 CFUs/cm2)

> PCL0.50-UA (105 CFUs/cm2) > PPO0.65-UA (106

CFUs/cm2)> PPO0.50-UA (107 CFUs/cm2).

(106)

45

Table 8 – Use of SLIPS as antifouling strategies with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Va

scu

lar

PTFE membrane

infused with

Perfluoropoly-

ether

Teflon®

(PTFE)

Porous PTFE; Fluoro-

silanized silicon; SLIPS

Fluorescence

microscopy; Crystal

Violet absorbance

P. aeruginosa

PA14, S. aureus

SC01 and E. coli

ZK2686

96% to 99.6% reduction on bacterial adhesion. (100)

Tethered liquid

perfluorocarbon

coating

PVC

PET TLP-PVC; TLP-PET

SEM; Crystal Violet

staining; Number of

CFU x 103

P. aeruginosa and

E. coli (ATCC

8739)

8-fold reduction in P. aeruginosa biofilm

formation after 6.5 weeks. (107)

Table 9 – Use of Sharklet Topography as antifouling strategy with possible application in catheters

Strategy

Catheter

material

Materials used on

strategy

Methods used to test

the strategy

Tested

microorganisms Results Ref.

Ty

pe

of

cath

eter

Uri

na

ry

Sharklet

micropattern Silicone SK; ISK; SK10x2

SEM; Number of

CFUs/cm2

E. coli (ATCC

#700336)

Reduction of the size of E. coli colonies in ≈ 76%

to 80%. (112)

Va

scu

lar

Sharklet

micropattern PDMS Sharklet AF™ PDMS

SEM;

Spectrophotometry

for adherent bacteria

S. aureus (ATCC

29213)

7% coverage by S. aureus after 14 days. No early-

stage biofilm formation until day 21. (109)

Sharklet

micropattern Polyurethane -3SK2x2; +3SK2x2

Number of

CFUs/cm2;

Fluorescence

microscopy; LSM

S. aureus (ATCC

6538) and S.

epidermidis (ATCC

35984)

Reduction of S. aureus and S. epidermidis adhesion

in ≈ 70%. (111)

46

5 Discussion

The development of catheters-associated medical infections starts with the attachment

of bacteria and subsequent biofilm formation on the surface of these biomedical devices.

Therefore, it is easy to understand why the prevention of bacterial adhesion is so important

(1,6,9,14,100,115).

Several antifouling strategies have been investigated through the years and some of

those were addressed in this work. In all of the reviewed strategies the authors presented

antibiofilm effective results and most of them have seem to be suitable for future applications

on biomedical devices in general and, in some cases, on catheters.

PEG coatings are known for proved antifouling properties. This polymer and derivatives

are the most used ones on medical devices, including catheters, for showing advantages such

as hydrophilicity, non-toxicity and biocompatibility (1,15,36,38,66,115). PEG has the ability to

form a water barrier – through hydrogen bonding with water molecules – that reduces bacterial

attachment (29,37,40). However, it is suitable to oxidative degradation in vivo, which reduces

the ability to resist to bacterial adhesion and limits its use for long-term applications (up to 14

days) (1,7,15,66,115).

For that reason, many researchers have made great efforts over the last decades to find

other alternative antifouling materials that are more stable than PEG but equally (or even more)

effective (15,36,38,66).

One of those alternatives are zwitterionic polymers (35,46,88). Similarly to PEG,

zwitterions are also able to form a hydrated layer on the surfaces but through strongly ionic

bonds. This leads to a more stable, biocompatible, dense and thicker barrier that makes

zwitterions better antifouling materials than PEG (1,18,35,80).

Beyond these ionic bonds, and specifically in case of SB (an example of zwitterionic

polymer), the number of water molecules that each unit of polymer can bind is also important.

In fact, each unit of SB is able to bind eight water molecules, comparing to one water molecule

in case of PEG (15,80). This may be another reason for the best antiadhesive properties of

zwitterionic polymers.

Concerning the use of naturally occurring molecules to antifouling applications,

polysaccharides are an attractive option. Of all the reviewed polysaccharides, DS, Hep, AG,

and MeCe were the ones that have demonstrated a higher resistance to bacterial adhesion and

47

biofilm formation (1,15,54), with results of bacterial adhesion inhibition > 90 %

(24,56,58,68,69). Also, they present good flexibility and biocompatibility, which makes of

these polymers interesting to biomedical applications (48–50).

PAAm is another polymer that has revealed to be a promising alternative to PEG

coatings. In fact, additionally to its good biocompatibility, PAAm has also demonstrated to

possess antiadhesive properties against some Gram positive and Gram negative bacteria as good

or even better than PEG (66,78,80,81).

Another interesting candidate to the development of other antifouling materials are

SLIPS surfaces. This original antifouling strategy based on Nepenthes pitcher plant has shown

a higher antifouling performance comparatively to PEG (about 35 times better) (100). Besides,

compared to SB coatings, SLIPS surfaces demonstrated to reduce bacterial adhesion and

biofilm formation for a longer period of time (107).

Sharklet microtopography (another naturally occurring-based antifouling strategy) is

also a promising alternative to the combat of bacterial adhesion. Sharklet has revealed to be a

great strategy with very good antiadhesive and antibiofilm properties. Also, it represents an

alternative to the use of chemical antifouling methods, since it uses topographical modifications

to resist to bacterial attachment (through induction of mechanical stress) (15,35,110,112).

Although the good antifouling results demonstrated by the reviewed studies, only a

small amount of them has been conducted under in vivo conditions (28,58,93). In fact, the most

part of these studies was developed under in vitro conditions, which are very different from in

vivo conditions.

So, given all that has been said so far, it becomes obvious how difficult it is to compare the

different strategies and choose one that stands out from the rest. This is related to the use of

different experimental conditions on the evaluation of the strategies. Examples of that are the

use of different bacteria types and strains, different inoculum loads, different controls and the

use of diverse methods that do not allow an effective quantification of adherent microorganisms

(e.g. SEM, crystal violet staining). Therefore, the use of a standard method should be

implemented as well as the use of methods that quantify the real number of adherent bacteria

like CFUs that are always more reliable in terms of perception of biofilm formation.

48

6 Conclusion

Given the constant growing in antibiotics resistance that has been verified in the current

years, it is essential to reduce the use of these molecules towards the prevention of HAIs. In

that way, it is crucial to act on the first step of the infectious process, i.e. bacterial adhesion.

Over the last few decades, many antifouling strategies have been developed and some

of them were reviewed in these work, namely the use of PEG, polysaccharides, PAAm and

polyacrylates, zwitterionic polymers, amphiphilic polymers, modified PUs, SLIPS and Sharklet

Topography to coat the surface of biomaterials.

All the reviewed strategies have been pointed by the authors to be effective in preventing

bacterial adhesion. Some of the best examples are zwitterionic polymers (more specifically

SBSi, PDA/PMEN10 and Poly-SB coatings), Amino-PPX-PAAm brushes, MeCe grafting and

associations of polysaccharides (Hep with CH or AG) that have demonstrated at least 93%

reduction of bacteria adherence. SLIPS and Sharklet Topography have also demonstrated

excellent antiadhesive and antibiofilm properties. In fact, a recent clinical trial revealed the

efficacy of Sharklet catheters on prevention of CAUTIs. So, these innovative approaches have

also proven to be promising candidates in the field of antifouling strategies.

Despite the large number of studies developed in the past decades, most of them were

conducted on PU, PDMS or silicone samples, aiming to evaluate the potential application of

the tested strategies on the field of biomedical devices.

Taking this into account, and given the current lack of studies specifically conducted on

catheters, it is necessary to develop more studies on those medical devices. Additionally, more

assays under in vivo conditions should be performed and presented.

Finally, a step towards the design of clinical trials, to get more answers for specific

questions about these strategies and to evaluate at the same time safety and efficacy, should be

taken.

49

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