ABSTRACT

Aim:

In this study, a new polymer gel dosimeter (PGD) was prepared using gamma rays for process validation of medical device sterilization. New polymeric systems were studied, consisting of itaconic acid, acrylamide, gelatin, different dyes [methylene blue, methyl orange and crystal violet (CV)] and N,N’-methylenebisacrylamide, with ascorbic acid as an oxygen scavenger.

Materials and Methods:

The study investigated the effect of the different dyes in the newly prepared PGD dosimeter formulations on the dose rate constants and radiation chemical yields in the high dosage ranging from 7 to 28 kGy-typical for the sterilization of many medical devices, pharmaceutical products and biological tissue-by using a ultraviolet-visible spectrophotometer method.

Results:

The sample spectrums of the prepared PGD dosimeters underwent a change following gamma irradiation, and bleaching of the blue gel color increased with the radiation dose. Even at the sterilization dose for medical devices (25 kGy), the dark blue color of the non-irradiated CV-PGD became almost colorless. The response of non-irradiated and irradiated PGD dosimeters remained stable during a storage period of 60 days.

Conclusion:

The prepared dye-PGD dosimeters are therefore suitable for use in monitoring various high dose radiation-processing applications, which can be useful for many medical devices and healthcare products.

INTRODUCTION

In the medical sector, process validation of medical device sterilization is crucial. Unfortunately, surgical devices are not properly sterilized in many places across the world. Many patients die or are diagnosed with various infections after surgery because of improper sterilization.

Authorities require regular sterilization as a mandatory process for the most of the medical equipments and devices¹. Sterilization is a process that ensures the medical device is without bacteria and any microorganisms. Medical devices are mostly reused for other patients after surgical operations, where they are contaminated by microorganisms. Therefore, effective sterilization is necessary in order to reuse such medical devices again. Ethylene oxide (ETO) sterilization, autoclaving as steam sterilization, radiation, hydrogen peroxide sterilization and chlorine dioxide sterilization are various methods of sterilization^1,2. To sterilize the medical devices such as syringes, implants, medical dressings, blood-bags and artificial joints methods such as gamma radiation from cobalt-60 (⁶⁰Co), X-rays or electron beams were used for many years. Sterilization of homogeneous systems and heterogeneous systems such as solid materials, gas materials, liquid materials and also medical devices are done gamma rays³. Gamma irradiation has physical inhibation effect on bacterial division by breaking the DNA of bacteria for providing decontamination to kill bacteria⁴. Contamination is caused by the energy of gamma rays passing through the equipments which disrupts pathogens. The reproduction capability or life of organisims causing contamination is finished by molecular changes induced by photons. Irradiation by gamma creates no radioactivity for medical equipment. Comparing with other methods of sterilisation gamma irradiation have reasons for preferance. Comparing with aseptic process method and filtration method, no excess ETO residues, advantage of less temperature for processing and easy validation of the sterilisation can be counted as the advantages of gamma irradiation⁵.

Eventhough dose is accepted as sterilisation standard parameter for validation of the sterilisation of medical equipment,to measure the dose is not an easy task. If the reponse to the dose of radiation is known and can be reproducibly achieved, then small blocks made of plastic, films, pellets or fluids may be used as dosimeters⁶.

Polymer gel dosimeters (PGD) are prepared from radiation-sensitive chemicals. These chemicals polymerize depending on the absorbed radiation dose^7-10. These gel dosimeters do not have recording limitations and maintain distribution in three-dimensions. They also have specific advantages when compared to one-dimensional dosimeters, such as ion chambers, and two-dimensional dosimeters, such as film.

Different PGD compositions and monomers in different types were studied. The aim was to create dosimetry system with temporal stability, spatial stability, dose-response exhibition in an optimal level, dose rate dependency and energy rate dependency for suitable and easy applications in clinic^11-13.

Tetrakis phosphonium chloride (THPC) or an oxygen inhibiter like ascorbic acid is homogenously used in these systems as an agent for crosslinking in an aqueous gel matrix. The changes in the physical properties of the material is produced by the reactions of crosslinking agents and monomers to initiate the polymerization and gel form from water radiolysis the formation of free radicals which are induced by ionizing radiation.

In this study, a new polymeric system consisting of itaconic acid (ITA)¹⁴, acrylamide (AAm), gelatin, different dyes [methylene blue (MB), methyl orange (MO) and crystal violet (CV)] and N,N’-methylenebisacrylamide (BIS), with ascorbic acid as an oxygen scavenger was studied. The use of doses from 0 to 1000 Gy have been already studied for hydrogel formation with the monomers ITA and BIS in an aqueous gelatin solution with THPC as an oxygen scavenger¹⁵. The effects of the different dyes in the newly prepared PGD dosimeter formulations were investigated in a high dose range from 7 to 28 kGy, typical for syringe sterilization, using a ultraviolet (UV)-visible spectrophotometer method.

GİRİŞ

Medikal sektörde, tıbbi cihaz sterilizasyonunun proses doğrulaması çok önemlidir. Ne yazık ki, cerrahi cihazlar dünyanın birçok yerinde uygun şekilde sterilize edilmiyor. Birçok hasta, uygun olmayan sterilizasyon nedeniyle kaybedilmekte ve ameliyattan sonra çeşitli enfeksiyon tanısı almaktadırlar.

Sterilizasyon, tıbbi cihazların çoğu için zorunlu olan ve düzenleyici otoritelerin gerekli kıldığı bir işlemdir¹. Cihazın bakteri ve diğer mikroorganizmalardan arındırılmasını sağlayan bir işlemdir. Tıbbi cihazlar sıklıkla yeniden kullanılır, ancak ameliyattan sonra mikroorganizmalar tarafından kontamine olur. Bu nedenle, bu tür tıbbi cihazların yeniden kullanılması için etkili sterilizasyon gereklidir. Otoklavlama, radyasyon, etilen oksit (ETO) sterilizasyonu, klor dioksit sterilizasyonu, hidrojen peroksit sterilizasyonu vb. gibi çeşitli sterilizasyon yöntemleri vardır^1,2.

İmplantlar, yapay eklemler, şırıngalar, kan torbaları, prematüre bebek üniteleri için biberon başlıkları ve pansumanların tamamı radyasyon kullanılarak sterilize etmek için Kobalt-60’dan (⁶⁰Co) gelen gama radyasyonu, elektron ışınları ve X-ışınları gibi metotlar yıllardır kullanılmaktadır. Gama ışınları genellikle gazlı, sıvı ve katı malzemelerin yanı sıra homojen ve heterojen sistemlerin ve şırıngalar, iğneler, kanüller gibi tıbbi cihazların sterilizasyonu için kullanılır³. Gama ışınlaması, bakteriyel DNA’yı parçalamak suretiyle bakteriyel bölünmeyi engelleyerek bakterileri öldürdüğü için fiziksel bir dekontaminasyon aracıdır⁴. Gama ışını enerjisi, kontaminasyona neden olan patojenleri bozarak tıbbi ekipmanlardan geçer. Moleküler düzeyde foton kaynaklı bu değişiklikler, kontamine edici organizmaların ölümüne neden olur veya bu tür organizmaları çoğalamaz hale getirir. Gama ışınlama işlemi, işlenmiş tıbbi ekipman üzerinde kalıntı oluşturmaz veya radyoaktivite sağlamaz. Gama ışınlamasının, steril ekipman üretmek için diğer yöntemlere göre bazı önemli avantajları vardır. Bu faydalar arasında filtrasyon ve aseptik işlemeden daha iyi ürün sterilliği güvencesi sağlaması, ETO gibi geride hiçbir kalıntı kalmaması, E-ışın işlemeden daha nüfuz edici, daha düşük sıcaklıklı bir işlem ve daha basit bir doğrulama süreci olması yer almaktadır⁵.

Tüm sterilizasyon standartları, ekipmanın steril olup olmadığını belirlemek için dozu anahtar bir parametre olarak kabul eder. Bununla birlikte, dozun ölçülmesi önemsiz bir görev değildir ve ticari dozimetri sistemleri, dozimetreler, okuma ekipmanı ve bunların kullanımına yönelik bir prosedürden oluşur. Dozimetreler, film, küçük plastik bloklar, sıvılar veya radyasyon dozuna⁶ bilinen ve tekrarlanabilir bir yanıt olan peletler olabilir.

Polimer jel dozimetreler (PGD), radyasyona duyarlı kimyasallardan hazırlanır. Bu kimyasallar absorbe edilen radyasyon dozuna bağlı olarak polimerize olur^7-10. Bu jel dozimetrelerin kayıt sınırlamaları yoktur ve üç boyutlu dağılımı sağlarlar. İyon odaları gibi tek boyutlu dozimetreler ve film gibi iki boyutlu dozimetreler ile karşılaştırıldığında özel avantajları da vardır.

Optimal bir doz tepkisi, geçici ve uzamsal stabilite, enerji ve doz hızı bağımlılığı sergileyen ve klinik uygulamalar için kolay ve uygun hazırlama yöntemleri sergileyen bir dozimetrik sistem hazırlamak için PGD için farklı monomerler ve bileşimler üzerinde çalışılmıştır^11-13.

Bu sistemlerde, çapraz bağlama maddeleri ve -çoğu durumda- askorbik asit veya tetrakis fosfonyum klorür (THPC) gibi bir oksijen inhibitörü sulu bir jel matris içinde homojen bir şekilde çözülür. İyonlaştırıcı radyasyon, monomerler ve çapraz bağlama maddeleri arasında polimerizasyon ve jel oluşumu reaksiyonlarını başlatmak için su radyolizinden serbest radikallerin oluşumuna neden olur ve böylece malzemenin fiziksel özelliklerinde değişiklikler meydana getirir.

Bu çalışmada, oksijen tutucu olarak askorbik asit ile itakonik asit (ITA)¹⁴, akrilamid (AAm), jelatin, farklı boyalar [metilen mavisi (MM), metil turuncusu (MO) ve kristal mor (KM)] ve N, N’-metilenbisakrilamid (BIS) içeren yeni bir polimerik sistem çalışılmıştır. Oksijen tutucu olarak THPC ile sulu bir jelatin çözeltisindeki monomer ITA ve BIS, 0 ila 1000 Gy arasındaki dozlar kullanılarak hidrojel oluşumu için çalışılmıştır¹⁵. Yeni hazırlanan PGD dozimetre formülasyonlarındaki farklı boyaların etkileri ultraviyole (UV)-visible spektrofotometre yöntemi kullanılarak şırınga sterilizasyonu için tipik olan 7 ila 28 kGy doz aralığında araştırılmıştır.

MATERIALS AND METHODS

Materials

AAm (99%), ITA (99%), BIS, ascorbic acid, MO and CV were obtained from the Sigma Aldrich Chemical Company. MB was supplied by Merck (cat no: 1,05045,0100). All the reagents mentioned above were used as received.

Method

All applicable international and national ethical guidelines were followed. No animal or human subject requiring ethics committee approval was included in the study. The optical absorbency of all irradiated samples was measured by a Shimadzu UV-visible spectrophotometer (Shimadzu UV-2401). The absorbencies of the PGDs were determined to be at wavelengths of 592, 664 and 462 nm before and 24 hours after their irradiation. These have been given as the ideal stabilization durationof the polymerization reactions within a PGD¹⁶. The optical absorbance of the irradiated sample (Ai) and its sample non-irradiated correspondence(Ao) difference is defined as Relative absorbance (∆A). Then, ∆A fits to a linear function of the dose (D), and PGD sensitivity is represented by the slope(s)¹².

∆A: Ai - Ao= sD + n                                             Equation (1)

The pH was measured using a pH meter (WTW pH 315i). The chemical characteristics of the PGD dosimeters were characterized using fourier transform infrared spectroscopy (Bruker VERTEX 70 ATR).

Preparation of the Polymer Gel Dosimeters

AAm-based gels were prepared based on the Venning method¹⁶ using 89% w/w of ultrapure deionized water, 5% w/w of gelatin, 3% w/w of BIS, 3% w/w of AAm, 1% w/w of ITA, 0.1% w/w of three different dyes (MB, MO and CV) and 10 mM of ascorbic acid. The gelatin was mixed with 90% of the water in dosimeters for a duration of ten minutes at room temperature. Then, the temperature was set to 45 ºC and constantly stirred to obtain the homogeneousity of the solution. Afterwards, BIS was added to the solution and mixed for 15 minutes at 45 ºC. After that, the temperature was decreased to 37 ºC and the AAm, IA and MB were added. The total solution was mixed at 37 ºC for 30 minutes and ascorbic acid was mixed with the remaining 10% of the water at 35 ºC. For two minutes the solution was kept in same condition. The prepared solutions were then put into glass tubes with stoppers¹⁷. For stabilization purposes, the dosimeters put in storage at 4 ºC for 24 hours before irradiation. Irradiation of all solutions was performed with a Nordion-Canada model JS 9600 model gamma irradiator from Gamma-Pak Ind & Trade Inc under air at 25 ºC. The PGD dosimeters were irradiated up to the maximum 25 kGy dose at a dose rate of 3 kGy/h.

Statistical Analysis

Equation 2 may be used to express the dye removal with gamma irradiation¹⁸. The constant of dose, k, is the natural logarithm (ln) of the slope of the compound concentration versus the absorbed dose.

ln (C/C0) = kD                                                             Equation (2)

where C is the concentration after gamma irradiation (M), C0 is the initial concentration (M), k is the dose constant (Gy-1) and D is the absorbed dose (Gy).

Necessary doses for Degradation percentage 50%, 90% and 99% degradation of CV (D_0.5, D_0.9 and D_0.99 values) constants were calculated by using equations 3, 4 and 5, respectively¹⁸.

D_0.5 = ln (2)/k                                                                Equation (3)

D_0.9 = ln (10)/k                                                            Equation (4)

D_0.99 = ln (100)/k                                                         Equation (5)

Table 1 shows the calculated k, and the D_0.5, D_0.9 and D_0.99 values for the different dye-PGD dosimeters prepared.

Absorbtion of 100 eV energy by degraded molecules is the definition of the G value¹⁹.

RESULTS

The G value was calculated using equation 6.²⁰

G=6.023x10²³ΔR/6.24x10¹⁷ D                               Equation (6)

where D is the absorbed dose (Gy), the dye (M) concentration change is ΔR, the factor of conversion from Gy to 100 eV/L is 6.24x10¹⁶ and the constant of avogadro is 6.023x10²³.

Table 2 shows the calculated G values for the different dye-PGD dosimeters prepared. The G values were observed to be in a continuously decreasing trend when the absorbed dose increased from 7000 to 28000 kGy for all different dye-PGD dosimeters.

The decrease in G values may have been related to the situation as the dye concentration decreases, where the dose absorbed increases (Figure 1)²¹.

DISCUSSION

Gamma irradiation is the most popular form of radiation sterilization and is used when materials are sensitive to the high temperature of autoclaving but are compatible with ionizing radiation²². Exposure is achieved when the packages are transported around an exposed ⁶⁰Co source for a defined period of time.

The European standard (EN 522) for the use of gamma rays on medical devices at a minimum dose of 25 kGy ensures the sterility assurance level of 10^-6. The international and European standards for the validation and routine control of medical device sterilization using ionizing radiation requires that a sterilization dose of 25 kGy should be effective²³.

PGD dosimeters were irradiated in a range from 7 kGy to 28 kGy. As shown in Figure 2, as the radiation dose increases, the color of the various PGD dosimeters is bleached. Even at a sterilization dose of 25 kGy, the dark blue color of the non-irradiated CV-PGD became almost colorless.

Three different PGD dosimeters containing different dyes (CV-PGD, MB-PGD and MO-PGD) were analyzed at their own absorbance peaks of 592, 664 and 462 nm wavelengths by an UV spectrophotometer. Figure 3 shows the calibration lines for CV, MB and MO, respectively. According to equation 1, the dose response of three different PGD dosimeters are summarized in Table 3 and Figure 4.

Study Limitations

The findings of this study should be evaluated considering some limitations. There is very little prior research on our specific topic, and we had to develop research typology. Dye-PGD dosimeters are suitable for use in monitoring various high dose radiation-processing applications and the usage in medical devices, pharmaceutical products and biological tissues should be supported with new studies for further development in this area.

TARTIŞMA

Gama ışınlaması, radyasyon sterilizasyonunun en popüler şeklidir ve malzemeler yüksek otoklavlama sıcaklığına duyarlı olduğunda, ancak iyonlaştırıcı radyasyonla uyumlu olduğunda kullanılır²². Maruz kalma, paketler belirli bir süre boyunca açıkta kalan bir ⁶⁰Co kaynağı etrafında taşındığında elde edilir.

Gama ışınlarının tıbbi cihazlarda minimum 25 kGy dozunda kullanımına yönelik Avrupa standardı (EN 522), 10^-6 sterilite güvence düzeyini sağlar. İyonlaştırıcı radyasyon kullanılarak yapılan cihaz sterilizasyonu, 25 kGy’lik bir sterilizasyon dozunun etkili olmasını gerektirir²³.

PGD ​​dozimetreleri, 7 kGy ila 28 kGy aralığında ışınlanmıştır. Şekil 2’de gösterildiği gibi, radyasyon dozu arttıkça çeşitli PGD dozimetrelerinin rengi ağartılır. 25 kGy’lik bir sterilizasyon dozunda bile, ışınlanmamış KM-PGD’nin koyu mavi rengi neredeyse renksiz hale geldi.

Farklı boyalar (KM-PGD, MM-PGD ve MO-PGD) içeren üç farklı PGD dozimetresi, bir UV spektrofotometre ile 592, 664 ve 462 nm dalga boylarındaki kendi absorbans zirvelerinde analiz edildi. Şekil 3, sırasıyla KM, MM ve MO için kalibrasyon çizgilerini göstermektedir. Denklem 1’e göre, üç farklı PGD dozimetresinin doz tepkisi Tablo 3 ve Şekil 4’te özetlenmiştir.

Çalışmanın Kısıtlılıkları

Bu çalışmanın bulguları bazı sınırlılıklar göz önünde bulundurularak değerlendirilmelidir. Spesifik konumuzla ilgili çok az ön araştırma bulunmakta ve araştırma tipolojisi geliştirmemiz gerekiyordu. Boya-PGD dozimetreleri, çeşitli yüksek doz radyasyon işleme uygulamalarının izlenmesinde kullanıma uygundur ve bu alanda daha fazla ilerleme için tıbbi cihazlarda, farmasötik ürünlerde ve biyolojik dokularda kullanım yeni çalışmalarla desteklenmelidir.

CONCLUSION

Sterilization of medical devices is very important in medical sector. Many different health risks arise in the absence of sterilization for health care products and surgical materials. To keep patients safe during the surgical process, medical conditions are extremely important during surgery. Thus medical sterilization is vitally important. Radiation sterilization has been widely used worldwide for the sterilization of health care products.

In this study, a novel PGD based on ITA, AAm, gelatin and different dyes (MB, MO and CV) was prepared. The sample spectrums of the prepared PGD dosimeters underwent a change following gamma irradiation, and bleaching of the blue gel color increased with the radiation dose. Even at the normal sterilization dose for medical devices (25 kGy), the dark blue color of the non-irradiated CV-PGD became almost colorless. The response of non-irradiated and irradiated PGD dosimeters was stable during a storage period of 60 days. Therefore, the prepared dye-PGD dosimeters are suitable for use in monitoring various high dose radiation-processing applications, which can be useful for many medical devices, pharmaceutical products and biological tissues.