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Wide Eyed

The topical administration of drops is an efficient and common delivery method for medicines of the eye. Patients who have undergone eye surgery need to self-administer drops to prevent or treat infections and inflammation in the weeks following their procedure. Moreover, a significant number of patients have conditions that require the long-term daily use of eye drops. For example, dry eye syndrome is associated with ageing, contact lens use, and environmental factors such as windy or sunny weather. It affects an estimated 5% of over 50s in the US, and is usually managed though the use of an artificial tear solution that needs to be applied between four to six times a day, and often for the rest of the patient’s life (1,2). Conditions such as hay fever and glaucoma also require the long-term use of self-administered eye drops.

It is important that all eye drops are kept free from bacteria. The microbial contamination of eye drops is a significant risk factor in the development of bacterial keratitis (3-8). Post-operative patients are at particular risk of infection, as are patients who have used topical steroids, since they lower the ocular defences (9).

Preservative-Free Formulations

One way of keeping multi-dose eye droppers safe is to add preservatives to the formulation. However, the use of preservatives can cause allergies or ocular irritation, and potentially a toxic response that may be damaging to patients’ eyes (10). Any such reactions are particular issues for those who rely on long-term use of eye drops for chronic conditions.

In 2009, the EMA stated that: “The inclusion of antimicrobial preservatives or antioxidants in a finished product needs special justification.” Even when preservatives are tolerated in adults, there are still questions over tolerance for the paediatric population. The EMA has stopped short of a general recommendation not to use preservatives in eye drops, but have recommended that “preservative-free formulations whenever possible should be considered” and “ophthalmic preparations without preservatives are strongly recommended for use in paediatric patients, especially neonates”. It has also noted that “it is nearly always technically possible to re-think the product development to remove [preservatives] or minimise their use” (11).

Single-dose eye droppers are a commonly used delivery method for preservative-free eye drops. By virtue of being applicable once only, there is no opportunity for bacterial contamination at point of use, meaning they are ideal for clinical settings – especially during surgery. However, they are costly and bulky, making them unsuitable for home use treatment of chronic conditions. Single unit preservative-free drops have been calculated to be 1,169% more expensive to produce than the equivalent preserved eye drops in a multi-dose bottle (12).

Intelligent Design


An alternative way to keep eye droppers clean is through the intelligent use of technology. Rather than relying on the anti-microbial properties of preservatives to kill any bacteria inside the bottle, the ideal approach is to prevent any bacteria from entering in the first place.

Multi-dose bottles dispense drops using either a non-return valve or a filtering system. Most commercially available bottles designed for multidose preservative-free eye drops rely on a filtering system to stop the entry of bacteria. When a drop is dispensed, the volume of the dose is compensated by air. Eye drops can therefore become contaminated in two main ways: by contaminated air entering the device, or by contaminated liquid reentering through the fi lter.

Filtering Out Bacteria

Anti-microbial filters are typically made of a membrane that consists of tightly packed layers of nylon fibre strands. Filters work on the mechanical principle that bacteria are large molecules that do not fit through the very small holes, while air and non-viscous solutions are able to pass without hindrance.

While 0.22μm sterile filters are the industry standard, their effectiveness as bacterial filters has been challenged in recent studies. It has been found that bacteria are capable of routinely penetrating 0.22μm filters, even when the molecules seem to be too big to fit through the holes.

In their 2002 paper, Wainwright et al found that: “Common, potentially pathogenic, bacteria (which are nominally larger than 0.2μm) can cross a 0.2μm nylon membrane. All of the bacteria crossed from the upper membrane surface to the solid medium below the membrane; this ability was highly repeatable and did not depend on the make of membrane used. Bacteria growing below the membrane exhibited normal size and morphology” (13).

In 2010, Onyango et al went further: “The assumption that filters with pore sizes less than 0.45μm can retain bacterial populations has repeatedly been disproved with the observation of regular passage of cells through 0.45μm, 0.22μm and even 0.1μm sterile filters.” Their findings showed that: “staphylococcal bacteria were capable of passing through sterile filters in a viable state” (14).

Even when filter effectiveness is shown, the bacteria clearly remain on the filter. A 2006 study on the efficacy of single-use bacterial filters revealed “a significantly greater bacterial growth on the proximal side of the filter compared with the distal side” (15). Eye drop filters act in two directions: pressure on the sides of the plastic bottle dispenses a dose through the filter to the patient’s eye; when the pressure is released, air and a small amount of liquid passes back through the filter and into the bottle. Therefore, bacterial growth on the filter represents a contamination risk for the delivered dose.

Questions of Unreliability

The evidence that bacteria can pass through 0.2μm filters is clear, but the reasons why are not obvious. One possibility for the observed passage of bacteria through filters could be due to the nature of the filter material itself. The sponge-like structure includes holes of varying sizes, some of which are statistically likely to be larger than 0.2μm. In fact, one study found that 0.2μm filters have a distribution of pore sizes that include some as big as 0.5μm (16).

Individual testing would eliminate doubt over the viability of each filter; however, the process used is destructive (17). The testing method introduces bacteria and liquid onto the filter surface. This starts bacterial growth on the filter and decreases the subsequent shelf-life of the dispenser. Therefore, in-line testing of multi-dose dispensers that rely on filter technology is not possible. Instead, testing is carried out statistically on only a proportion of the dispensers.

In addition, it is likely that there are other mechanisms of bacterial penetration. Hasegawa et al found that the bacteria Pseudomonas aeruginosa “passed through a 0.22μm pore size filter. The membranes which allowed passing-through of bacteria showed normal bubble point values in the integrity test” (18). This demonstrates that bacteria are still capable of passing through a reliable 0.22μm pore size filter.

The study by Onyango et al refers to the finding of 0.5μm holes in a 0.2μm filter, but notes that this “is highly unlikely the reason for the observed result in our study”. Instead, they point to bacterial motility as a major factor in filter penetration (14,16).

Bacterial Motility


Bacteria come in a variety of shapes and sizes, although many of them share the ability to self-propel by twitching, rotating or gliding. Twitching is the most common form of motility and is achieved through movement of the flagella in a way that makes the bacteria appear to swim. Studies have shown that this motility enables bacteria to move through very small channels relative to their size.

Hasegawa et al demonstrated that P. aeruginosa were able to pass through a reliable 0.22μm pore size filter. They then experimented with a strain of the bacteria that was defective in twitching motility and found that it was unable to pass through the same filter (18). It was concluded that it is the flagellum-dependent motility of P. aeruginosa that enables it to penetrate fine filters.

Meanwhile, in their 2009 paper, Männik et al set out to establish how the relatively large Escherichia coli and Bacillus subtilis bacteria can move in very narrow channels. They found that: “both E. coli and B. subtilis are motile in channels which only marginally exceed their diameters [by ~30%]” (19). Furthermore, the E. coli lose their ability to swim in channels narrower than their diameter. Despite this, however, they are still able to enter narrow channels. Over time, it was observed that through the mechanism of growth and division, E. coli bacteria were able to penetrate filter channels “with a width that is smaller than their diameter by a factor of approximately two. Within these channels, bacteria are considerably squeezed, but they still grow and divide.”

This has clear implications for the effectiveness of filters for multi-dose preservative-free eye droppers. Filtering liquid several times a day means that the filter remains wet throughout the usable lifetime of the device, presenting ideal conditions for bacterial growth.

Alternative Solution

A viable alternative to the use of sterile filters for multi-dose preservative-free eye droppers is a non-return valve system used in conjunction with a silicone membrane to filter the returning air. The one-way valve ensures that no contaminated liquid can be reintroduced to the container after the drop has been dispensed, completely removing the need to filter the liquid. The intake of air into the dispenser takes place via a separate venting system with a silicone membrane.

The venting system filters the intake of air using a very fine membrane manufactured from silicone polymer. This membrane is a solid, non-porous material; it is homogenous and does not contain any holes. Its characteristics can, therefore, be precisely engineered. The membrane’s intermolecular distance is of the order of nanometers, allowing the passage of air through the membrane, but completely preventing the passing of any liquid or solid, including bacteria.

The function of the membrane can be compared to an inflated balloon. The balloon is a continuous, waterproof material, yet gas slowly passes through the wall of the balloon until the pressures inside and outside reach equilibrium. In the same way, the separation of the dose delivery from the venting system means that the membrane is kept dry. This minimises the risk of bacterial growth on the membrane surface, and also means that the testing process is non-destructive. In fact, devices that use this technology can be tested individually in-line as a consistent part of the manufacturing process to ensure robust quality standards, providing an even greater assurance of safety for patients.

One-Way Progress

A non-return valve combined with a silicone membrane venting system demonstrates how intelligent design can be used to prevent the entry of bacteria into a bottle, making it possible to deliver safe, multi-dose preservative-free eye drops.

References
1. Schaumberg DA, Sullivan DA, Buring JE and Dana MR, Prevalence of dry eye syndrome among US women, Am J Ophthalmol 136: pp318-326, 2003
2. Schaumberg DA, Dana MR, Buring JE and Sullivan DA, Prevalence of dry eye disease among US men: Estimates from the Physicians’ Health Studies, Arch Ophthalmol 127: pp763-368, 2009
3. Templeton WC et al, Serratia keratitis transmitted by contaminated eyedroppers, Am J Ophthalmol 93(6): pp723-726, 1982
4. McCulloch JC, Origin and pathogenicity of Pyocyanea in conjunctival sac, Arch Ophthalmol 29: pp924-936, 1943
5. Hogan MJ, The preparation and sterilization of ophthalmic solutions, Calif Med 71: pp414-416, 1949
6. Theodore FH, Contamination of eye solutions, Am J Ophthalmol 34: p1,764, 1951
7. Theodore FH, Practical suggestions for the preparation and maintenance of sterile ophthalmic solutions, Am J Ophthalmol 35: pp656-659, 1952
8. Vaugh DG Jr, The contamination of fluorescein solutions with special reference to Pseudomonas aeruginosa (Bacillus pyocyaneus), Am J Ophthalmol 39: pp55-61, 1955
9. Rahman MQ, Tejwani D, Wilson JA, Butcher I, and Ramaesh K, Microbial contamination of preservative free eye drops in multiple application containers, Br J Ophthalmol 90(2): pp139-141, 2006
10. Report of the International Dry Eye Workshop, Ocul Surf 5(2): pp65-204, 2007
11. EMEA Public Statement on Antimicrobial Preservatives in Ophthalmic Preparations for Human Use. Visit: www.ema.europa.eu/docs/en_GB/document_library/Presentation/2010/09/WC500096784.pdf
12. Hertel F and Pfeiffer N, Einzeldosisapplikationen in der glaukomtherapie, vergleich der kosten mit mehrdosis, Ophthalmologe 91: pp602-605, 1994
13. Wainwright M et al, Big bacteria pass through very small holes, Med Hypotheses 58(6): pp558-560, 2002
14. Onyango LA, Dunstan RH and Roberts TK, Filterability of staphylococcal species through membrane filters following application of stressors, BMC Research Notes 3: p152, 2010
15. Unstead M, Stearn MD, Cramer D, Chadwick MV and Wilson R, An audit into the efficiency of singleuse bacterial/viral filters for the prevention of equipment contamination during lung function assessment, Respir Med 100: pp946-950, 2006
16. Osumi M, Yamada N and Toya M, Bacterial retention mechanisms of membrane filters, J Pharm Sci Technol 50: pp30-34, 1996
17. Benezech T, A method for assessing the bacterial retention ability of hydrophobic membrane filters, Trends in Food Science & Technology 12: pp36-38, 2001
18. Hasegawa H, Naganuma K, Nakagawa Y and Matsuyama T, Membrane filter (pore size, 0.22-0.45μm; thickness, 150μm) passing-through activity of Pseudomonas aeruginosa and other bacterial species with indigenous infiltration ability, FEMS Microbiology Letters 223: pp41-46, 2003
19. Männik J, Driessen R, Galajda P, Keymer JE and Dekker C, Bacterial growth and motility in sub-micron constrictions, PNAS 106(35): pp14,861-14,866, 2009


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Katriona Scoffin is a freelance science writer and marketing consultant for Nemera and other drug delivery solution providers. She has extensive experience writing for life sciences, including contract research, drug discovery, instrumentation, delivery devices, labware and software. Katriona has a degree in Physics and works in Cambridge, UK.
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