The pH values of microstructures solutions were measured with a Schott TitroLine by simply plunging the electrode into the PU aqueous solutions (1:5000 v/v). The samples present slightly acid pH values (6.61 for sample PU_1, 6.13 for sample PU_2, and 6.44 for sample PU_3) due to the characteristics of its components. The absence of secondary products (amines) is demonstrated by the weak acid character of suspensions. Beside, these pH values are appropriate for products intended for cutaneous administration [12
The synthesis of different hollow polymer microstructures for drug delivery field is nowadays a research topic of high interest [13
]. The aim of this study was the synthesis and testing of PU microstructures with low toxicity and controlled-sized particles. For drug delivery systems small size particles are very important in order to overcome the defence barrier of corneous layer [15
], but on the other hand the quantity of encapsulated drug must be taken into consideration. In the PU microstructures synthesis the presence of an initiator to form the chains is not necessary. In this case the assembly of polymer chains and the formation of the polymer shell take place simultaneously, thus it is called an in-situ
polymerization method [17
]. In principle, the method can be adopted in systems where the monomer is soluble in water while the corresponding polymer is insoluble in water at the reaction conditions.
During scanning electron microscopy (SEM) investigations, the existence of microstructure aggregates of irregular shapes was detected (Figures , and ); the shape and the size are not influenced by the diisocyanate used. The microstructures are not spherical in shape which consequently leads to a poor flowing ability. The morphology of these PU agglomerates is not characteristic for a porous-type material which represents an advantage for an intended transdermal vehicle in order to protect its load. Non-agglomerated microstructures cannot be successfully synthesized probably due to their low mass.
SEM images of PU1 microstructures.
SEM images of PU_2 microstructures.
SEM images of PU_3 microstructures.
Differential scanning calorimetry (DSC) showed that the degradation is due to a thermooxidative process (exothermic effect) and takes place at temperatures significant higher than the melting point of the polymer (Figures , and ). As observed, the initial temperatures of the thermooxidative process were closed, as follows: PU_1 (297°C) > PU_3 (281°C) > PU_2 (276°C). The final temperatures were: 292 and 296°C for PU_2 and PU_3, respectively. Also, one can notice the absence of glass transition temperature which is determined for most polymers; its measurement depends on a crystalline transition so its absence usually reveals the predominant amorphous nature of a polymer [18
The thermal decomposition behaviour of PU_1 microstructures.
The thermal decomposition behaviour of PU_2 microstructures.
The thermal decomposition behaviour of PU_3 microstructures.
The Zetasizer results (Table ) show large values for the diameter of each sample (over 500 nm) which could be attributed to the particle aggregation process. The zeta potential values are significant because if all the particles have a zeta potential more negative than −30 mV or more positive than +30 mV the dispersion should remain stable [19
]. All this considered, the product obtained in the second experiment, when isophorone diisocyanate (IPDI) was used, is regarded as the most stable.
The Zetasizer characterization for the PU microstructures
Noxiousness investigations were carried out on mesenchymal stem cells; sources of stem cells described in the literature are: bone marrow, peripheric blood, citaferesis concentrate, umbilical cord and placenta [20
]. The main source for MSCs and haematopoetic cells remains bone marrow which was also used in our experiments.
All experiments were done in quadruplicate so the absorbance average of four wells was calculated. Values thus obtained were introduced into the following formula in order to calculate the reduction level of Alamar Blue reagent by metabolic activity of cells:
where AB%N is the percentage of reduction of Alamar Blue reagent compared to the negative control (medium without cells); O1 represent molar extinction coefficient (E) for oxidized Alamar Blue at 570 nm; O2 is molar extinction coefficient (E) for oxidized Alamar Blue at 600 nm; R1 is molar extinction coefficient (E) for reduced Alamar Blue (red) to 570 nm; R2 is molar extinction coefficient (E) for reduced Alamar Blue to 600 nm; A1 represent absorbance of tested cells at 570 nm; A2 is absorbance for tested cells at 600 nm; N1 is absorbance of negative control (medium with Alamar Blue and without cells) to 570 nm; N2 is absorbance of negative control (medium with Alamar Blue and without cells) to 600 nm [21
]. The results are expressed as the mean for each quadruplicate culture ± the standard error.
A high percentage of reduced Alamar Blue (red) reactive indicates a strong metabolic activity and consequently an increased viability and cell proliferation, while unviable cells produce an oxidized medium (blue). Previous studies made on silicon-based microparticles have shown that surface properties and chemistry may influence the adhesion and function of cells in culture [22
All samples exhibited metabolic activity as measured by the detected fluorescence of the Alamar Blue reagent after 2 hours incubation at 37°C. Alamar Blue results were significantly different between day 1 and 2 (Figures and ), but cells were still viable throughout the experiment.
The MSCs viability after 24 hours.
The MSCs viability after 48 hours.
A good result for MSCs viability was recorded for PU_1 after one day, but we consider MSCs viability after 48 hours to be more important. In this case, the best value was recorded in case of PU microstructures based on IPDI. The main conclusion Alamar Blue test produced was the reduced noxiousness for the studied polymeric microstructures.
CD1Nu/Nu (nude mice) present an abnormal hair growth, due to functional follicles but failed hair growth and that is why the skin gives a quick response to any external aggressive factor. This mice type was chosen for our experiment because their skin has the following important features: it is very sensitive and exhibits a penetration degree a few times higher than human skin [23
]. The sensitivity of these mice’ skin is an advantage as it can be used as a parameter characterizing the investigational product noxiousness. In this study, Tween®20 and diisocyanates by their specific group (−N = C = O) are known as dangerous substances for human health [25
], but on the other hand we have demonstrated above that the polyurethane products did not reveal any toxicity. In the present work an excess of polyol was used in order to avoid the diisocyanates toxicity issue, excess easily removed by washing and useful to prevent formation of secondary amine products.
The transepidermal water loss (TEWL) for the hairless mice was evaluated by non-invasive techniques for the characterization of skin changes [27
Because the application of a new topical compound could determine important skin parameter changes (e.g. erythema), the assessment of local haemoglobin and secondary melanin (for pigmented animals) content could be helpful. The Courage-Khazaka instruments are manufactured to be generally used in cosmetic tests on human skin. This is the reason why, previous to this research, we created a database (unpublished) with mouse skin parameter values (measured on batches consisting of four different species of mice, of various ages, with and without skin lesions). In this study, the evolution of TEWL values for the four CD1Nu/Nu mice batches (one blank group and three groups corresponding to the applied PU microstructures) lies in the range 0–10 g/h/m2
which is specific to skin in very good condition (Figure ) [28
]. The mice batch where PU_2 was applied shows a stationary trend over the four weeks so one can conclude that IPDI based PU microstructures are appropriate ingredients for skin application. In other cases TEWL values slightly increase indicating a reduced damage. Increased TEWL values indicates a skin dehydration process [29
] but if the values are not very high above the limit increased TEWL values do not indicate a certain sign of noxiousness.
TEWL evolution ± SEM: TEWL, pH, melanin, and erythema.
The measured pH average values for CD1Nu/Nu mice skin are between 6 and 8. One can notice in Figure that batches 2 and 3 (where PU_2 and PU_3 were applied) least change their values during the experiment. Measured pH values indicated no important changes at skin level [30
] leading to the conclusion that application of these PU formulations maintained the pH of skin.
The erythema, measured by the Mexameter®MX 18 probe, is a parameter which slightly increases its values during different skin testing [31
]. When erythema values change rapidly and significantly it usually is an indicator of an important skin stress such as skin injury, infection or inflammation [32
]. In this experiment the lowest change of the erythema (local haemoglobin) values was in case of mice batch 2 (where PU_2 was applied) which recommends this sample as a transdermal carrier with the most reduced noxiousness Figures and .