Calcium oxalate crystallization in synthetic urinary medium: the impact of resorcinares and calixarenes†

Odin Bottrill , Matthew Boon , Franca Jones * and Mauro Mocerino
School of Molecular and Life Sciences, Curtin University, Kent Street, 6152, Perth, Western Australia, Australia. E-mail: F.Jones@curtin.edu.au

First published on 20th January 2022

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Introduction

Biomineralisation is an evolutionary response by organisms utilised for a number of purposes, ranging from formation of skeletons to gravitaxis.¹ However, biomineralisation can also be pathological; for example, leading to the uncontrolled formation of kidney stones. In the case of kidney stones, calcium oxalate is known to be the main species (70% of cases).² Kidney stones are mainly made of whewellite or calcium oxalate monohydrate (COM, CaC₂O₄·H₂O) and/or whedellite or calcium oxalate dihydrate (COD, CaC₂O₄·(2 + x)H₂O with x ≤ 0.5), though this hydrate is less stable in the medium.³ There is also calcium oxalate trihydrate or caoxite (COT, CaC₂O₄·(3 − x)H₂O with x < 0.5), although this is not observed due to its very low thermodynamic stability in the medium.

The pathology of kidney stones brings into question how can this process be controlled or mitigated? A field of research focused around this is that of crystal growth modifiers. Crystal growth modifier is a general term for a species that can alter some aspect of the crystal's formation. Crystal growth modifiers are chemicals (that can be referred to as additives) that can affect the crystal size, shape or nucleation rate of particles. One possible mechanism of modification is through the chelation of one of the crystal's constituents in solution, lowering the supersaturation. Alternatively, the additive could bind to the surface of the crystal, resulting in the slowing or prevention of growth at the binding sight, effecting the morphology.⁴ Once adsorbed to the surface the additive can also effect the zeta potential around the particle and thereby impact the possibility of aggregation.⁵ Additives could also stabilise other polymorphs or hydrates through methods such as templating.⁶ Templating is a method whereby a large molecule interacts with the crystal, forming an interface that directs and controls the crystal's formation and can cause selective formation of hydrates and polymorphs.⁷ Incorporation of the additive is also possible, where the additive is internal to the crystal resulting in an altering in the crystal's behaviour and growth. There has already been some research into the use of crystal growth modifiers for the control of the formation of kidney stones,^8–21 although many of the studies investigate the impact in simple solutions unrelated to real life conditions.

In the case of kidney stone formation in urine some aspects of the development of stones are well explored. Generally, the accepted mechanism of stone formation begins with the supersaturation of cation and anion that then undergoes nucleation. The crystal then interacts with the cells in the kidneys, holding the stone in place and allowing the stone to grow larger over time.²² During the formation of the stone there are a large number of organic species ranging from small organics to proteins present in the urine which can interact with or incorporate into the crystal. Analysis of calcium oxalate stones has shown the crystal's internal matrix can consist of a number of other smaller molecules and proteins.²³ It is still contentious in the literature if these proteins play a role in the inhibition or promotion or are simply incorporated during the crystallisation phase.

There are some options for patients for the removal of kidney stones. However, treatment of the condition as a re-occurring disease is largely absent. There is some interest in the possible treatment of the condition using citric acid (or derivatives) and phosphates from the literature.²⁴ Additionally, it is recommended patients who are reoccurring stone formers undergo dietary changes to prevent the intake of the relevant species causing the condition. Due to the limited treatment options for reoccurring stone formers new chemicals must be tested and researched as possible therapeutics to prevent or inhibit the continued formation of these stones. Much of the research associated with additives is restricted to smaller molecules (or some identified proteins in the urine) and little has been done with larger molecular additives.

Calixarenes have been widely used for many purposes from medicinal to imaging.^25–28 The reason for the variety of uses is due to the amount of freedom the base structure has for functionalisation (Fig. 1a). Another advantage is within the circular structure itself, as it keeps the functional groups in a well-defined location, for example, with groups all on the wider rim of the circular structure and at known distances. This not only aids in the interpretation of results but also allows the functional groups to have strong interactions as the presence of the functional groups on one side of the scaffold can lead to more favourable binding between the molecule and the crystal surface.

There has been much research in terms of the synthesis of novel calixarenes, although many of these only get tested for few purposes.²⁹ The aim of this research is to observe the impact of calixarenes and resorcinarenes on calcium oxalate crystallization and to guide this avenue of research for the future (and ultimately to new therapeutics). This work expands on earlier research on possible inhibitors using smaller molecules using the same synthetic urinary medium (SUM) and methods³⁰ whilst also expanding on research into calixarenes as crystal growth modifiers.^28,31 More specifically, this work will look at the functionalisation of these base structures with amino acids (aspartic acid, lysine and proline) and observe the impact that the molecule has on the formation of calcium oxalate nucleation and formation. The impact of the molecules will then be compared to the relevant amino acid to see the impact that having those amino acids attached to the large organic scaffold has on the observed crystals. In addition, the impact of zinc ions was also investigated. Zinc ions are normally present in urine and have been found to have a complex impact on calcium oxalate crystallisation.³² Finally, it should be stressed that even this SUM is not a true synthetic urine but is closer to reality than matching the ionic strength with NaCl due to the presence of commonly found ions such as magnesium and phosphate.

Materials and methods

Materials

All reagents were of analytical grade quality, or they were used as synthesised. The water present or added in all the experiments and stock solutions was deionised water. A stock solution of the SUM was prepared at 10 times the concentration as reported in a previous paper, shown in Table 1, and adjusted to a pH of 6.5 with hydrochloric acid.³⁰ Potassium phosphate monobasic was stored separately to avoid phosphate precipitation occurring in the SUM. To avoid bacterial growth in the SUM, sodium azide (1% w/w) was added. CAUTION: sodium azide is hazardous and should be handled according to Safety Data Sheet instructions.

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Zinc ions are also present in urine and as such the interaction of zinc ions with SUM was tested by adding zinc chloride. Thus, the SUM with zinc ions is a more physiologically comparable SUM. The zinc chloride solution stock solution (0.1 M) was kept separate from the SUM solutions and was added into the medium such that the final concentration was 1 mM, which is within the normal range for human urine.³⁴ Each of the organic inhibitors investigated is shown in Table 2. The synthesis procedure and characterisation of the calixarenes and resorcinarene molecules can be found in the ESI.†

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Morphological tests

A 20 mL solution was prepared by using deionised water, 2 mL of the SUM stock solution and 2 mL of phosphate monobasic stock solution. Sodium oxalate solution 0.4 mL, 0.1 M was then added. A clean glass disk of 0.5 cm radius was added to the vial to allow for analysis of the solids formed. The additive (Table 2) was then added at the desired concentration. The calixarenes and resorcinarenes were tested at concentrations of 1 mM while the amino acids were tested at 4 mM to account for the macrocycles containing 4 equivalents of the amino acid in the structure. Deionised water was added, if required, to ensure that the final volume was 20 mL for all experiments. The mixture in the vial was left for 15 to 20 minutes until the temperature equilibrated to 37 °C before adding 0.4 mL, 0.1 M of calcium chloride to commence the crystallization process. This solution was left for 18 to 24 hours after which the glass disk was collected, washed gently with water, and dried.

In the cases where zinc ions were added to the medium, zinc chloride was present at a final concentration of 1 mM for all tests and added prior to the calcium chloride addition. This concentration was chosen as it is within the normal concentration range for human urine.³⁴

The recovered glass disks were then analysed by Raman spectrometry using a WITec alpha 300SAR (Bruker) to determine the hydrate form. The Raman functionality utilises a frequency-doubled NdYAG laser of wavelength 532 nm (green) of 50 mW power with silicon being used as the reference material.

After observing the effects of the additives on the crystals using the optical microscope and Raman spectroscopy, the disks were placed on carbon coated scanning electron microscope (SEM) stubs and carbon paint was applied to the circumference to avoid charging effects. The SEM stubs were sputtered with platinum to a thickness of 2–20 nm and analysed with a Neon focused ion beam – SEM (Zeiss) operating at 15 keV with a secondary electron detector.

Crystal size measurements

The particles on the glass disk were analysed by optical microscopy and images were taken to measure the area of COM crystals. Measurements were only undertaken in cases where the whole crystal was unobscured by other crystals. For all of the crystals meeting this criteria, regardless of size, a minimum of 60 crystals from each optical image were measured giving the area and the aspect ratio (length/width). The aspect ratio was found to have a 10% variance. This analysis was performed using the software ImageJ®.

Analysis of nucleation rate and zeta potential

The determination of particle counts versus time through dynamic light scattering was used to assess the impact of additives on the nucleation rate. Similarly, the propensity of particles to aggregate was inferred by way of the magnitude of the measured zeta potential.

A 19.2 mL SUM solution was prepared by combining filtered water and the urinary medium (2 mL), followed by potassium phosphate (2 mL, 32.3 mM), zinc chloride (if required) and/or additive stock (depending on the test) solution, water was added if required to make to the final volume 20 mL. Sodium oxalate (0.4 mL, 0.1 M) was then added in a Teflon beaker and stirred constantly. An aliquot of the solution (approximately 2 mL) was placed in a disposable cell and analysed using the Zetasizer Nano-ZS (Malvern) at 25 °C. Measurements were recorded using a DPSS laser with an excitation wavelength 633 nm. The standard operating procedure was set with an integration time of 10 seconds, taking 10 measurements. After recording the baseline ‘counts’ of the solution, stoichiometric calcium chloride was added, and a measurement was undertaken every 5 minutes. The aliquot of the solution that was removed for these analyses was then returned to the solution. This was then repeated until 20 minutes had passed (where nucleation was typically observed to peak). For each experiment, the time and the derived counts (in kilocounts per second, kcps) was recorded. Each experiment was repeated in triplicate and the presented data are the average of the three measurements with error bars associated with the standard deviation of these tests.

Finally, 25 minutes after the addition of the calcium chloride, zeta potential measurements were taken from the same experiment. These tests use a folded capillary cell, containing ∼1 mL of the slurry. The instrument collects a maximum of 100 measurements and records the value only when the maximum number of measurements or concordant values are obtained. The above sequence was repeated in triplicate and the values were averaged to provide a final measurement for each additive.

Raman depth analysis

The organic molecules tested may adsorb or incorporate into the COM or COD crystals. The Raman spectroscopic instrument using a frequency-doubled NdYAG laser of wavelength 532 nm (green) of 50 mW can be focussed at different heights. Scanning through the crystals allows investigation of the signal response for the additives within the crystal. The entire particle was analysed by focusing the laser at different depths, whereby line scans were undertaken creating layers of signals that overlay to form a heatmap of signals across the z and y axis. From the heatmap the position of signals throughout the crystals can be examined which can elucidate mechanisms of inhibition of promotion. In addition, the Raman spectrum can be diagnostic of either COD or COM (where the ∼1500 cm⁻¹ peak is a doublet for COM and a singlet for COD).

Results and discussion

Nucleation

Crystal morphology

Within SUM, calcium oxalate monohydrate (COM, Fig. 8a) was exclusively observed. The morphology of the COM formed within the SUM has rounded edges (see Fig. S1a†), through the inhibition of the (100), (021) and (121) faces, which results in a more circular morphology. This is due to the presence of citric acid in the SUM which is a well-known inhibitor of CaOx.^23,24 COD could be stabilised in the presence of additives and the standard morphology is shown in Fig. 8b.

Additional SEM images showing the particles formed in the presence of the additives with and without zinc ions can be found in the ESI† section (Fig. S1 and S2†).

Crystal size and aspect ratio

In Tables 3 and 4 the comparison between the impacts of the amino acid functionalised macrocycles and their associated amino acids on the particle sizes can be seen. It is important to note that, where possible, the crystals measured were the individual crystals not the aggregates of the crystals.

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The area of the formed crystals in the presence of aspartic acid is slightly higher than the standard, although within experimental error. The particles formed in the presence of Lys are approximately half the size of those formed in the control with those formed in the presence of Pro smaller still. The presence of the PRAsp forms COD particles and so strictly speaking the size cannot be compared to the standard or to the Asp system. Having said this, the COD particles formed in the presence of PRAsp are much smaller than the COM particles formed in the presence of Asp. The presence of CPro does form significantly smaller particles than the control although not as small as proline alone. In contrast the presence of PCLys formed even smaller particles than Lys alone.

The smaller particle size observed in the case of PRAsp could also be due to the nucleation promotion observed, but nucleation promotion is also observed in the case of Asp and the final particle sizes are larger than the standard particles. This suggests that the presence of Asp promotes growth of COM as well as nucleation. When the Pro and CPro are present the particle counts (and hence nucleation rate) are close to the standard, but the sizes of COM particles is much smaller than the standard suggesting that growth of COM is inhibited by these additives. In the case of Lys and PCLys, nucleation was mildly promoted but the growth is significantly impacted as seen in the SEM images and the size data.

While the PRAsp could not be investigated with zinc ions present because the crystals could not be recovered from the solution the respective amino acid was still tested.

When observing the impact on size (for the case of SUM + Zn²⁺) the presence of the amino acids always resulted in particles which were smaller than the control with Asp forming the smallest particles for the amino acids. The particles formed in the presence of PCLys were, however, the smallest of all when zinc ions were present and significantly smaller than those of the control. For Asp the smaller particle sizes could be due to the increased nucleation, however, this cannot be the case for proline. In this case, the smaller size must be due to growth inhibition. In addition, the extent of promotion when Lys or PCLys are present cannot account for the small size that is observed when PCLys is present suggesting again that this impacts growth of COM significantly (Table 5).

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The particles formed in the presence of the free amino acids result in similar aspect ratios; the change in the aspect ratio being at most 0.09 (and within the 10% error). The effect of the amino acids alone seems minor, although the nucleation impact of these varies, with Pro being similar to the standard and Asp being a notable promoter. The aspect ratio of CaOx particles formed in the presence of the macrocycles is significantly altered. For the PRAsp case, this is because COD particles are formed while when CPro is present this highlights the elongation of the particles. In the case of PCLys there is only a slight elongation, and the dominant change is actually in the aggregate shape (Table 6).

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When comparing the effect the presence of zinc ions has on aspect ratio, again the amino acids all appear to have a similar impact. The largest difference in aspect ratio is 0.22 for the case of Asp and is the only significantly different result for the free amino acids. This means the particles formed are slightly longer than the control particles. The impact of PCLys on CaOx particles formed in the presence of zinc ions show a value of 1.44 which is larger than the control and suggests longer particles are formed. In this case, it should be noted, the aggregate shape is not being measured but any individual particles observed.

Incorporation of the additive

After analysis of the size and shape of the crystals the next step was to determine what could be occurring to give the response observed. To do this the crystals were analysed using Raman spectroscopy, where the focal point of the laser can be at different depths in the crystal giving multiple spectra for different layers through the crystals. Since different vibrational frequencies can be observed for the CaOx particles and the organic additive, this can provide a ‘heatmap’ for a particular signal (Fig. 14), which can be highlighted to show the strength of the signal throughout the crystal. The signals chosen are typically those which are different to standard COM or COD standard spectra and compared to the COM or COD signal present. In this way, a relative region where the additive is most concentrated can be determined.

The crystal analysed through depth analysis were required to be alone and showing the (100) basal face. For the COM crystals it was analysed from one (010) face to the other to keep the analysis consistent. For COD crystals the cross section analysis is across the (100) face without going through the (010) (ESI† Fig. S9–S11).

Aggregation potential

Zeta potential measures the double layer around a particle and, at low ionic strength, is related to the surface charge. More importantly, the zeta potential can give information on the likelihood of aggregation, as particles in a solution with similar surface charge should repel each other. However, it is found that generally particles with zeta potentials <±30 mV are likely to aggregate.⁵ Thus, zeta potential can give information on the likelihood of larger stone formation through an aggregative process.

The effect of PRAsp on the resulting zeta potential (Fig. 22) is significantly beyond the effect of any other additive recorded in these tests. The high absolute magnitude of the zeta potential reduces the likelihood of aggregation to form larger particles. The high absolute magnitude will mean the lowest likelihood of aggregation of the particles in the presence of this molecule. In addition to charge stabilisation, these larger molecules may also sterically stabilise particles against aggregation, thus a lower zeta potential may not be as critical for these macrocyclic additives. It should be noted that the PRAsp formed COD particles and that based on these results the COD is less likely to aggregate than the COM particles formed under the tested conditions.

This is in stark contrast to the presence of Asp which shows particles with a zeta potential closer to zero. This is likely due to the tendency for the molecule to form a zwitterion at the experimental pH. Due to the smaller magnitude of the zeta potential, when present as an additive, Asp will likely increase the tendency of particles to aggregate. This shows that the interaction is being made by the combination of the base resorcin[4]arene structure and the functionalised chemical, which leads to a significant effect on the zeta potential that is much stronger than just the Asp.

The particles formed in the presence of CPro show a zeta potential close to zero. The reason for the low zeta potential could be due to the CPro only containing four carboxylic acid groups as opposed to the 8 present in PRAsp. Alternatively, the presence of a phenolic hydroxide at the narrower rim of the structure may cause some additional interaction resulting in this lower magnitude. As the phenolic hydroxyl is neutral the presence of these groups in the double layer may result in a smaller absolute value of the zeta potential. However, the Pro amino acid results in COM particles with a larger magnitude of zeta potential. Thus, the macrocyclic scaffold is impacting on this value.

The impact the presence of Lys has on zeta potential is significantly different to the other additives, displaying a positive zeta potential where the other molecules resulted in CaOx particles with a negative or slightly negative value. Also of note is the impact of the PCLys, which shows a significant increase from the amino acid response. This is similar to the impact seen with the PRAsp. In the presence of PCLys this increase means that the particles are less likely to aggregate as the magnitude is greater than 15 mV. This highly positive impact is likely due to the total absence of any carboxyl acid moiety as this molecule is functionalised though the carboxylic acid using an amide bond, leaving two free primary amines. These can then interact with the charge around the particle giving this highly positive result. This in itself is interesting due to the low concentration (1 mM) of additive present compared to the other species in the SUM.

It is interesting to also note that the impact of the additives on zeta potential appears to have some relationship with the isoelectric points of the free amino acids at least in the absence of zinc ions.

It can be seen (Fig. 22 and 23) that in comparison to the standard, zinc ions increase the zeta potential in magnitude but make the particle more negative. This is despite the addition of the positive ions of zinc. One possible way in which this may occur is if the zinc ions adsorb to the surface and then attract the negative counter-ions from the SUM to the particle resulting in a more negative value.

Zinc ions in the solution results in minimal change in the zeta potential of the particles formed for the CPro and Pro case, which do not result in a significantly lowered magnitude. PCLys experiences no significant change in the presence of zinc ions either, which is likely due to the molecule being positively charged and as such there is no likely interaction between zinc ions and the calixarene. However, the presence of Asp results in a notable increase (more positive value) in the zeta potential. When both PRAsp and zinc ions are present in solution there is a significant change (∼8 mV) on the measured zeta potential. This may be due to the ability of PRAsp to chelate, which could result in some of the PRAsp interacting with zinc ions in solution resulting in a less negative zeta potential from the additive when zinc ions are present.

One of the most interesting impacts of zinc ions is the effect on the Lys amino acid, where the signal changes from ∼5 mV to ∼−2 mV which is the largest change present when zinc ions are introduced. This is likely due to some of the zinc ions forming complexes with lysine which has been observed previously.³⁷ This lowers the zeta potential of the CaOx particles but will result in the zeta potential still remaining closer to zero than the standard in SUM. The effect of chelation is not observed with PCLys, which is due to the fact that this chelation with the amino acid would occur through the carboxylic acid and primary amine site and the carboxylic acid is the functional group that is used in the coupling, making it unavailable for interaction. That is, for the PCLys, the chelation between the Zn²⁺ and the amino acid cannot occur since the amide bond was formed between the carboxylate of the amino acid and the calixarene, leaving only the amine functional groups free.

Conclusions

Calixarenes and resorcinarenes are promising scaffolds for investigation of the crystal growth modification of calcium oxalate. These amino acid functionalised molecules have shown that the large impact the additives have had on the nucleation rate, crystal growth and the zeta potential values of calcium oxalate particles formed is essentially due to the functionalisation onto the scaffold. Considering their overall interactions, the most interesting of the additives are both PCLys and PRAsp. These molecules result in calcium oxalate particles with very large zeta potentials (with and without the presence of zinc ions). This means that PCLys and PRAsp will likely have a significant impact on the aggregation of the particles – reducing this likelihood.

Additionally, PRAsp causes the stabilisation of COD in the SUM, which could make the formation of stones even more difficult. On a less positive note, the presence of PRAsp did show that nucleation is significantly promoted, however, more nucleation could lead to smaller particles that are less damaging. The presence of PCLys significantly alters the size of the crystals causing the formation of much smaller crystals. Also of note is that the crystal's morphology is very different with the faces being indistinguishable. The crystals could indicate the possibility of branching and appear to form roughly spherical aggregates. There is still much to learn about why these molecules have the impact they do but all of the additives tested showed incorporation into the crystal through the Raman depth analysis. This means that after adsorption, the additives are strongly interacting with the surface, so much so that the additive is then incorporated into the crystal. PRAsp being found less on the surface may suggest that most of this additive is already incorporated by the time the crystal is analysed.

Whilst CPro had no significant impact on the zeta potential or nucleation rates there was still a very notable impact on the morphology. The crystal form, both in the presence of the SUM and the SUM + zinc ions, show evidence of layering. Like the other additives, incorporation was also confirmed through Raman depth analysis and while the impact appears mainly on the growth processes, the interaction between additive and calcium oxalate particles is still strong enough for incorporation to occur.

Based on the analysis of all the data it seems that the additives are all likely adsorbing onto the surface of either the nuclei or the growing particles and interacting with the crystal throughout crystallisation. Using the depth analysis it can be identified that the signals for the organics are found to incorporate suggesting a very strong interaction. This strong adsorption mechanism would also explain the significant change in the size and morphology of the crystals formed as well as the significant impacts on the zeta potential.

The varied responses from the amino acid functionalised calixarenes and resorcinarenes opens up a large avenue for functionalisation and investigation of new molecules. It is especially interesting that the impact occurs despite there being very minimal impact on the crystals from the free amino acids. This may in future also give insights into the significant and varied impact of proteins. These results show crystal growth modification for calcium oxalate can be achieved at low concentrations. Thus, this data shows evidence that these molecules should be further investigated. In future, whether these macrocyclic molecules result in promising therapeutic treatments for reoccurring kidney stone formers will depend on much more research into their toxicity, bioavailability, in vitro and, finally, in vivo efficacy.

Author contributions

Using the CRediT taxonomy: OB conceptualised the project, conducted the investigation, formal analysis of data and wrote the first draft. MB collected SEM data (investigation). FJ and MM conceptualised the project, supervised and had scientific input into data interpretation (formal analysis) and writing of subsequent drafts.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We would like to acknowledge the John de Laeter Center and Curtin University facilities and equipment. We would also like to thank the Australian Government for funding via the Research Training Program scholarship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthetic methods, SEM images, optical images, Raman spectra. See DOI: 10.1039/d1ce01445e

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