# The in vivo fate of 225Ac daughter nuclides using polymersomes as a model carrier

## Abstract

Increasing attention is given to personalized tumour therapy, where α-emitters can potentially play an important role. Alpha particles are ideal for localized cell killing because of their high linear energy transfer and short ranges. However, upon the emission of an α particle the daughter nuclide experiences a recoil energy large enough to ensure decoupling from any chemical bond. These ‘free’ daughter nuclides are no longer targeted to the tumour and can accumulate in normal tissue. In this paper, we used polymersomes as model carrier to evaluate the retention of recoiling daughters of 225Ac in vivo, and assessed their suitability as therapeutic agents. Vesicles containing 225Ac were injected intravenously in healthy mice, and intratumourally in tumour-bearing mice, and the relocation of free 213Bi was assessed in different organs upon the injection [225Ac]Ac-polymersomes. The therapeutic effect of 225Ac-containing vesicles was studied upon intratumoural injection, where treatment groups experienced no tumour-related deaths over a 115 day period. While polymersomes containing 225Ac could be suitable agents for long-term irradiation of tumours without causing significant renal toxicity, there is still a significant re-distribution of daughter nuclides throughout the body, signifying the importance of careful evaluation of the effect of daughter nuclides in targeted alpha therapy.

## Introduction

Personalized medicine is a rapidly growing field in cancer therapy research. The use of tumour-specific therapeutics has proven to significantly increase patient survival and decrease side effects. A number of β-emitting radiotherapeutics are routinely being used for the treatment of e.g. metastasized prostate cancer and neuroendocrine tumours1,2. An increasingly popular alternative can be found in the use of α-emitting radionuclides. Only a few α particles passing through the nucleus are sufficient to cause multiple double-strand breaks in the DNA and subsequent cell death. Due to their much higher linear energy transfer (LET), α-emitters are more cytotoxic than β-emitters3. Other advantages of the use of α-emitters in tumour therapy include their independence on tumour oxygenation, and their short tissue range which prevents damage to neighbouring healthy cells.

Thus far, promising preclinical and clinical results have been reported for a number of α-emitters, including 213Bi (t1/2 = 45.6 min) which has successfully been used to treat a different tumours4,5. A lot of attention is currently being paid to 225Ac (t1/2 = 10 days), which provides clear advantages over 213Bi including a long half-life allowing more time for accumulation at the tumour site. Furthermore, 4 α particles are emitted in the decay chain of 225Ac, resulting in a larger dose to the tumour site per mother nuclide. A number of successes have been realized in clinical trials using 225Ac, including treatment of neuroendocrine tumours, prostate cancer, and gliomas5. The use of 225Ac-PSMA-617 for the treatment of metastatic castration-resistant prostate cancer shows some particularly promising results6. Even upon progressive disease during after 2 cycles of 177Lu-PSMA-617, impressive treatment response to treatment with 225Ac-PSMA-617 were observed7. However, one of the potential issues arising when using 225Ac for therapy is unwanted toxicity from recoiled daughter radionuclides. Upon the emission of an α particle the radioactive daughter nuclides experience a recoil energy of about 100–200 keV8 which is much larger than the energy of any chemical bond and will thus always result in the daughter nuclide breaking free from the targeting agent. Furthermore, the different chemical properties of the daughter nuclide can make re-association with the chelator very unlikely9. These ‘free’ daughter nuclides can be a source of dose limiting toxicity10. For instance, long-term renal toxicity ﻿has been observed in a study using 225Ac labelled anti-rat HER-2/neu monoclonal antibody due to the relocation of recoiling daughter atoms to the kidney11. In another study by Kennel et al., the potential of killing lung tumours in vivo was clearly demonstrated but at the cost of radiotoxic effects associated with released daughter radionuclides which were likely partially responsible for the death of the animals12.

Encapsulation of the 225Ac mother nuclide in nanocarriers can help retain the daughter atoms at the tumour site and thus limit damage to healthy organs. This approach has been investigated with a number of nanoparticles, including metal-based particles13, zeolites14 and liposomes15. Woodward et al. demonstrated that LaPO4 based nanoparticles are capable of containing 225Ac daughter nuclides. They observed a near complete retention of 225Ac in the nanoparticles, but found that about 50% of the daughter nuclides were released in vitro. Subsequently, they followed the in vivo release of 213Bi in the lungs, liver, spleen and kidney. Elevated levels of 213Bi were found in the kidneys, indicating some release of this daughter nuclide following nuclear recoil, despite its encapsulation in the nanoparticle16. 225Ac-containing liposomes have been shown to be very efficient at selectively killing tumour cells in vitro17. Their efficacy has been tested in 3D tumour models as well as in vivo, introducing a diffusion-assisted approach for full tumour coverage. Here, the liposomes degrade upon entering the lower-pH tumour environment, releasing 225Ac and its daughter nuclides and hence allowing for a better distribution in vitro. This study has shown promising in vivo results, with a significant decrease in tumour volume upon the intravenous administration of the liposomes18. The distribution of the daughter atoms in vivo was not assessed, nor was potential renal toxicity considered, however, very poor 213Bi retention in 100 nm liposomes has been reported in an earlier in vitro study19, suggesting similar problems to occur in vivo.

The aim of our study is to assess the retention of the radioactive daughter atom 213Bi in a carrier system which has demonstrated good retention of the 225Ac mother nuclide, and determine the in vivo fate of free 213Bi20. We have selected polymersomes as delivery vesicles for 225Ac-based targeted α therapy8,20,21, which have shown to retain the daughter nuclides 221Fr and 213Bi to a certain extent20. This retention can be improved by forming small InPO4 nanoparticles around the 225Ac encapsulated within the polymersomes21. Nanocarriers are known to accumulate in the liver and spleen, whereas free 213Bi accumulates in the kidneys, allowing for clearly distinguishable uptake characteristics between the polymersomes themselves and any free daughter nuclides. In this study, we evaluate the retention of 213Bi, one of the daughters in the decay chain of 225Ac, in vivo upon intravenous and intratumoural injection, and looked at the distribution of free 213Bi in select tissues, This is especially important to consider when using long-circulating carriers like nanoparticles or antibodies for tumour targeting. We assessed the effect of the polymersome diameter as well as the presence of InPO4 nanoparticles encapsulated within the vesicles on the retention and distribution of 213Bi. Finally, the effect of [225Ac]Ac-polymersomes and [225Ac]AcDOTA control on tumour growth, proliferation, apoptosis, and double strand DNA breaks upon intratumoural injection are evaluated in BALB/c nude mice.

## Results and Discussion

It is well known that the recoil energy experienced by the daughter atom is sufficient to break any chemical bond between the daughter atom and a targeting vector. This issue could partially be circumvented through the use of nanocarriers, such as polymersomes, which present a system which is capable of retaining at least part of the daughter nuclides. We have used polymersomes to encapsulate 225Ac in the aqueous cavity of the vesicles containing either DTPA20 or InPO4 nanoparticles21. In this study, we compared the recoil retention of the 213Bi daughter nuclide in vivo of both these polymersome formulations. Throughout this manuscript, ‘free 213Bi’ indicates 213Bi which is no longer encapsulated in the polymersomes following nuclear recoil.

Prior to using the polymersomes in the in vivo studies, they were fully characterized by both DLS and Cryo-TEM (Fig. 1). In all cases, relatively monodisperse particles were observed, with an average diameter of 97 ± 37 nm as measured by CryoTEM. As mentioned before by Wang et al., DLS measurements of the average diameter yield a slight overestimation compared to the CryoTEM images22. The InPO4 nanoparticles (18 ± 9 nm in diameter) could also be nicely visualized within the polymeric nanocarriers.

Polymersomes were loaded with 225Ac according to the well-documented procedures as described earlier by Wang et al.20 and de Kruijff et al.21. A loading efficiency of 54 ± 21% has been achieved in the DTPA-containing polymersomes, and 59 ± 6% for co-precipitation in InPO4 containing polymersomes. The labelling efficiency of the [225Ac]AcDOTA compound used for intratumoural injection was 92.3%.

### Recoil retention 213Bi

#### Intravenous injection

We assessed the two different polymersome formulations, 225Ac chelated with DTPA or 225Ac coprecipitated with an InPO4 nanoparticle, on the in vivo recoil retention of 213Bi upon intravenous injection. While the 225Ac mother nuclide is very well retained in both systems (retention of approximately 93%)20,21, this is not the case for the daughter radionuclides. The theoretical recoil distance of the daughter radionuclides is about 100 nm in water8, which means that the probability of the daughter nuclides to be being retained in polymersomes with an optimal diameter of around 100 nm is limited23,24. The use of larger polymersomes will increase the retention of daughter radionuclides, but this is known to reduce the circulation time and subsequent accumulation in tumour tissue21. Another factor influencing the retention of recoiled daughter is the location of the decaying radionuclide in the vesicle. For instance, if the mother nuclide 217At (second daughter of 225Ac) is located in the polymersome bilayer, the chance of the 213Bi daughter recoiling out of the vesicle is much higher than when 217At would be located in the centre. The loading of 225Ac into polymersomes through co-precipitation with InPO4 nanoparticles has been shown more successful in retaining daughter nuclides compared with 225Ac loaded through chelation with DTPA21. When 225Ac is co-precipitated with InPO4, the recoil distance decreases to about 30 nm, greatly increasing the probability of daughter retention.

To determine whether these predictions also hold within a more complex in vivo system, the distribution of 213Bi after administration of [225Ac]Ac-polymersomes was determined in mice. With a half-life of a little under an hour, the ingrowth of 213Bi was determined through continuous measurement of the ex-vivo organ activity in time. These measurements allowed for extrapolation to the time of sacrifice and a direct comparison of the presence of 213Bi and 225Ac at organ level (see Fig. 2).

Unfortunately, given both the short half-life of 221Fr (t1/2 = 4.8 min) and the time it took to perform the biodistributions together with the relatively large distance between the animal facility and gamma counter, we were not able to accurately assess the distribution of 221Fr. Like most nanocarriers, polymersomes accumulate mainly in the spleen and liver25, while free 213Bi is known to accumulate mainly in the kidneys (40%), or is excreted via the urine (30%)26. To be able to measure the 213Bi ingrowth with a certain degree of accuracy, it was important to keep the total number of samples small so that they could be measured frequently. To this end, it was decided to determine the recoil retention of 213Bi in three organs of interest; the blood (injection site), spleen (polymersome accumulation) and kidneys (accumulation of free 213Bi). Immediately upon sacrifice, the organs of interest were collected and continuously measured until the daughter nuclides were in equilibrium with the mother nuclide 225Ac. Back-extrapolation of the obtained data enabled the determination of the amount of 213Bi at the moment of sacrificing the animal, as this is the time point at which blood flow stops, stopping subsequent inter-organ redistribution of free daughters. The activity at this time ($${A}_{{213}_{Bi}}(t=0)$$) therefore represents the activity of 213Bi nuclides both from within the polymersome, as well as any redistributed free 213Bi. At equilibrium, the 213Bi activity ($${A}_{{213}_{Bi}}(t=eq)$$) comes only from the decay of 225Ac in the polymersomes, and thus gives an accurate representation of the biodistribution of the polymersomes. The ratio of these two numbers indicates the distribution of free 213Bi, and shows that the redistribution of daughter nuclides has a non-negligible effect on the dose distribution of α therapy to the different organs.

In Table 1, the activity of 213Bi at the moment of sacrifice (t = 0) as well as in equilibrium with 225Ac is given for selected organs. Furthermore, the ratio of the 213Bi at t = 0 to the equilibrium activity of 213Bi after in-growth to the mother nuclide activity is given. When this ratio is <1, it means that the organ of interest is releasing 213Bi (in this case the blood and spleen), while for a ratio >1, the organ is accumulating free 213Bi. The daughter nuclide distribution presented in Table 1 was measured 4 h after the injection of 225Ac encapsulated in either DTPA-containing polymersomes, or co-precipitated in InPO4 nanoparticles within the polymersomes. In both cases, a significant difference (p < 0.02) between initial 213Bi presence and 213Bi levels at equilibrium with 225Ac was found in the blood. Significantly more (p < 0.05) 213Bi was retained in the blood for the InPO4 containing vesicles vs the DTPA ones (with ratios of 0.14 ± 0.07 and 0.06 ± 0.03 respectively). Based on the blood values, co-precipitation of 225Ac with InPO4 nanoparticles in polymersomes constitutes a definite improvement over DTPA containing polymersomes. However, the ratio of the redistributed 213Bi at time of sacrifice to the equilibrium 213Bi activity found in the spleen and to the kidneys is not significantly different (at a significance level of α = 0.05) when considering the two different polymersome formulations. At first glance this is surprising; a lower amount of free 213Bi would be expected to be present in the mice injected with InPO4-containing polymersomes based on the blood values, which should have resulted in ratios closer to 1 for both the spleen and the kidneys. However, in the kidneys the accumulation of InPO4-containing polymersomes is nearly a factor two lower than for the DTPA-containing polymersomes (2.6 ± 1.1% ID/g vs 4.7 ± 0.8% ID/g respectively), which subsequently increases the ratio of free 213Bi to the equilibrium activity of 213Bi in polymersomes to similar levels. Free 213Bi present in the kidneys at time t = 0 nearly exclusively originates from the release of daughter nuclides from the polymersomes in other organs. The amount of free 213Bi in the kidneys at time of sacrifice therefore more accurately represents the amount of recoiled 213Bi than the ratio does. There is significantly less free 213Bi at t = 0 in the kidneys for the InPO4 containing polymersomes (at a significance level of α = 0.05) which agrees with the differences observed in the blood. For the spleen, similar ratios between the DTPA and InPO4 containing polymersomes were observed. This is most likely explained by the fact that macrophages within the spleen take up the polymersomes, and the macrophages themselves can than act as a secondary barrier against the escape of the 213Bi daughter nuclides from the macrophages. This effect has been described by McDevitt et al., who used 225Ac-labeled antibodies, and found that upon internalization in the cell, the cell aids in the retention of the daughter nuclides27.

In this study, we have assessed the retention of 213Bi in the polymersomes upon intravenous injection, and looked at the distribution of free 213Bi in organs of interest. However, whereas the daughter radionuclides are retained to at least some degree within the polymersomes themselves, they will always be released when chelated to a tumour-targeting antibody due to the recoil effect28, with amongst others the different chemical properties of the daughter nuclide making re-association very unlikely. Therefore, it is of great interest to compare the results obtained in our study to antibody-targeting studies assessing the recoil retention in vivo. Unfortunately, to date there are only very few studies looking at the free daughter distribution upon decay of 225Ac attached to an antibody. McDevitt et al. recognized that the retention of the daughter alpha-emitters at the target site is critical to the success of the therapy, and therefore specifically focussed on the use of target cell-internalized 225Ac constructs, where internalization by the tumour cell itself resulted in enhanced retention of the daughter nuclides27. The daughter nuclides 221Fr and 213Bi were very well retained at the tumour site at 2 days p.i. (88 ± 9% and 89 ± 2% respectively), although it has to be mentioned that these tumours were counted between 6 and 12 minutes after death and not extrapolated back to time of death, increasing the calculated tumour retention. Kidney values of 213Bi show a significant accumulation of free 213Bi, at least 3 times higher than at equilibrium. However, the equilibrium values of the 225Ac-antibody constructs in the kidneys are much higher compared to the low uptake of [225Ac]Ac-polymersomes found in our study, which significantly decreases the ratio of free 213Bi to that in equilibrium with 225Ac. Furthermore, their values were reported in cpm instead of % ID/g preventing the direct comparison of free 213Bi uptake. Another study by Jaggi et al. looked in detail at the distribution of 225Ac daughter nuclides upon the intravenous injection of 225Ac-labeled antibodies9. While in most cases they do not specifically assess the ratio of the free daughter nuclide to the equilibrium situation, they do show a kidney-to-femur ratio of 225Ac which is much lower than that of 213Bi (an estimated ratio of approximately 1 for 225Ac vs 20 for 213Bi) indicating significant redistribution of 213Bi to the kidneys. Song et al. used 225Ac-labeled anti-rat HER-2/neu monoclonal antibody and found a difference in kidney activity of 213Bi at time of sacrifice of 2449.8 Bq/g to 887.0 Bq/g at equilibrium11, also concluding that there is significant uptake of free 213Bi in the kidneys. Therefore, while all these studies show that there is indeed redistribution of free 213Bi, confirming the results obtained in our study, the presented ratios cannot be compared directly.

#### Intratumoural injection

To determine the distribution of free daughter nuclides after accumulation in the tumour, 225Ac-containing polymersomes were intratumourally injected and the distribution of 213Bi was quantified immediately upon sacrifice. In these experiments, 225Ac was encapsulated into polymersomes through chelation with DTPA. Although the polymersomes where 225Ac was co-precipitated with InPO4 yielded better 213Bi retention upon intravenous injection, polymersomes containing DTPA have been studied more extensively20,22, including their therapeutic potential in vitro29, and were thus selected for the intratumoural experiments presented in this paper. Again, free 213Bi was expected to be transported by the blood to the kidneys, and hence the organs of interest in this study were the blood, kidneys and tumour. Polymersomes with a diameter of either 100 nm or 200 nm were intratumorally injected to study the effect of polymersome diameter on daughter nuclide retention. These larger vesicles were not used in the intravenous study, as polymersomes should ideally have a diameter below 100 nm23 for optimal circulation times.

Table 2 shows the retention of 213Bi in the tumour tissue, giving both the amount of 213Bi present in tissue at time of sacrifice (t = 0) as well as at equilibrium. The ratios of free 213Bi to 213Bi in equilibrium are also displayed in this table, where again a ratio is <1 signifies a release of 213Bi in the tissue of interest, and a ratio >1 an accumulation of free 213Bi. Clearly, the tumour is in all cases the only tissue releasing free 213Bi, where the 213Bi is subsequently transported by the blood to the kidneys amongst others. However, it has to be kept in mind that the ratios for both the blood and the kidneys appear exceedingly high due to near-zero amount of polymersomes present in these organs at equilibrium. Rather, for these organs the activity of free 213Bi at time of sacrifice should be regarded as the main indicator of their free 213Bi accumulation. The very low radionuclide activity in the blood shows that 213Bi is transported by the blood but not retained there. There was no large difference in free 213Bi uptake between the 100 nm and 200 nm vesicles in the blood, although in all cases kidney uptake of free 213Bi was slightly lower for the 200 nm polymersomes. This corresponds to the retention of free 213Bi in the tumour tissue, where although already quite well retained for 100 nm polymersomes, 213Bi is nearly completely retained in the tumour when encapsulated in 200 nm vesicles. However, for both polymersome diameters, no significant increase in tumour retention of 213Bi is seen over time, which indicates that the size of the polymersomes still influences the retention of the daughter nuclides suggesting that part of the vesicles has not yet been taken up by the tumour cells. This corresponds to earlier obtained results in in vitro tumour spheroids, where the polymersomes were still distributing themselves throughout the spheroid more than 4 days after the addition of fluorescently labelled polymersomes to the cell medium29. Looking specifically at potential kidney toxicity, the activity of 213Bi at time of sacrifice is in all cases much higher in the tumour than in the kidneys, with tumour: kidney ratios of 9.9 for 100 nm polymersomes, and 29.5 for 200 nm polymersomes at 7 days p.i. Renal toxicity due to free 225Ac daughter atoms which have recoiled out of the polymersomes was thus not be expected to be a problem for the therapeutic studies20.

A study by Woodward et al., examined the recoil retention of 213Bi in LaPO4 nanoparticles containing 225Ac and showed that the release of 213Bi from the target organ decreased over the course of 5 days16. They attributed this to the potential uptake of their nanoparticle conjugates by the endothelial cells of the lungs, which subsequently trapped any free 213Bi within the cells, retarding its diffusion through the tissue. We did not observe a similar pattern in our study; the retention of 213Bi in the tumour tissue is stable in time for both polymersome sizes. This difference in behaviour could be due to the different injection methodologies. Woodward et al. injected their nanoparticles intravenously allowing them to be taken up in the target tissue in time. Although they did not present any direct measurements of circulation time of the nanoparticles, the activity in the target organs is still increasing up to 48 h p.i., pointing to a long circulation time during which the nanoparticles are not completely taken up in the endothelial cells yet. On the other hand, our polymersomes were injected directly within the tumour tissue and hence did not circulate for an extended period of time, resulting in no significant increase in retention between 1 and 7 days p.i.

### Biodistribution

#### Intravenous injection

While 221Fr emits gamma particles with energies suitable for SPECT imaging16, the activity used in this experiment is too low for proper imaging which is why we performed a biodistribution study. The distribution of 225Ac complexed to DTPA in polymersomes was compared to that of 225Ac co-precipitated with InPO4 nanoparticles in polymersomes upon intravenous injection. Biodistribution data of the [225Ac]Ac-polymersomes at 4 h p.i. can be seen in Fig. 3, for both 225Ac coupled to DTPA or encapsulated in InPO4 nanoparticles. Because the radionuclides are encapsulated within the aqueous core of the polymersomes, the outer surface fo the polymersomes remains the same, logically resulting in the similar organ uptake. The only organ displaying a significant difference in uptake between the two types of vesicles is the liver. At the moment, we have no good explanation for this difference in liver uptake. The two types of polymersomes (containing either InPO4 or DTPA in the aqueous cavity) were composed of the same batch of block copolymers, prepared according to the same method, and injected in mice which had been randomly distributed between the two groups. Since the outer surface of the polymersomes was not changed, a difference in uptake was not expected, and further research would be required to fully understand this difference in uptake. The circulation time of these polymersomes in tumour bearing mice is known to be considerably shorter than in healthy mice, with circulation half-lives of 5 min and 117 min respectively30. The exceedingly short circulation half-life in tumour bearing mice prevents sufficient polymersome accumulation at the tumour site upon the intravenous injection of 111In-containing polymersomes (0.44 ± 0.39% ID/g). We therefore decided to assess therapeutic efficacy of [225Ac]Ac-polymersomes through intratumoural injection.

#### Intratumoural injection

For the biodistribution and therapeutic study of intratumorally administered [225Ac]Ac-polymersomes, vesicles with a diameter of 100 nm were used. Here, the biodistribution of both intratumourally injected 225Ac complexed to DTPA in polymersomes as well as [225Ac]AcDOTA was assessed at 1 and 7 days p.i. The reason for the use of [225Ac]AcDOTA instead of 225Ac-DTPA as a control for the polymersomes is its increased kinetic stability31. [225Ac]AcDTPA has been shown to display substantial toxicity due to 225Ac leakage, whereas [225Ac]AcDOTA constitutes an substantial improvement31. While this lower stability of the [225Ac]AcDTPA complex has been shown not to result in enhanced 225Ac leakage from the polymersomes20, the more stable [225Ac]AcDOTA complex was chosen for the in vivo studies to minimize toxicity due to unbound 225Ac. In Fig. 4, the distribution of both [225Ac]Ac-polymersomes and [225Ac]AcDOTA in selected organs is displayed. When viewing these biodistribution results, it is important to keep in mind that the injected activity as well as volume were very small, due to which the activity measured in various organs was just slightly above background. As expected based on earlier results by Wang et al.25, the polymersomes are very well retained in the tumour tissue, while the [225Ac]AcDOTA compound is rapidly cleared. The tumour retention of the polymersomes was found to be 244 ± 74% ID/g and 289 ± 130% ID/g at 1 and 7 days p.i. respectively, whereas less than 10% ID/g and 5% ID/g of the [225Ac]AcDOTA was retained at the tumour site at both time-points respectively (Fig. 4).

Uptake in most other organs is minimal for both compounds. In the [225Ac]AcDOTA study, nearly no activity is found in any other organ, suggesting that the majority of the compound was excreted already at time of sacrifice. The only organ exhibiting some uptake of [225Ac]AcDOTA are the kidneys at 1 day p.i., indicating that renal excretion is indeed the main pathway for [225Ac]AcDOTA clearance. At 7 days p.i., renal uptake is present only in insignificant amounts. The [225Ac]Ac-polymersomes are much better retained by the tumour tissue than [225Ac]AcDOTA. Organs other than the tumour exhibiting uptake of [225Ac]Ac-polymersomes are the liver and spleen, and to a lesser extend also the bone and muscle. The main cause for healthy tissue uptake can likely be found in release of the polymersomes themselves. Polymersomes which have diffused out of the tumour tissue will either be filtered out by Kupffer cells, which are responsible for the phagocytic activity of the liver, or by macrophages in the spleen which corresponds well with the uptake observed in Fig. 432. The 225Ac presence in the healthy organs could also, in part, be caused by 225Ac which was released from the polymersomes. Both free 225Ac as well as chelated 225Ac have been shown to accumulate in the bones as well as in the liver31,33,34, and is temporarily also retained in the kidneys33,35. Davis et al. have shown that this kidney uptake is very time dependent and much smaller than the liver uptake, so even though kidney uptake in our study is minimal, there might be some free 225Ac responsible for the organ uptake. However, in earlier work, the 225Ac released from the polymersomes was found to be only ﻿2% of the total amount of encapsulated 225Ac over a period of 8 days, making the release of 225Ac from the polymersomes in vivo unlikely20. These biodistribution results indicate that renal toxicity from 225Ac-polymersome accumulation is unlikely to play a major role in inducing dose-limiting toxicity, though any health issues related to the kidneys and bone marrow should be carefully studied.

### Tumour growth and overall survival upon intratumoural injection

The tumour model chosen, MDA-MB-231, is a well-vascularized tumour model. Upon injection of the compounds (the polymersomes as well as the DOTA and PBS), some blood was observed to leak out of the tumours. In Fig. 5, the tumour growth of the 8 mice per group bearing subcutaneous MDA-MB-231 tumours is presented. At 28 days after inoculation the tumours were injected with the various compounds. In 7/8 tumours injected with [225Ac]Ac-polymersomes a complete inhibition of tumour growth was observed (Fig. 5A). Two out of eight tumours doubled in size (at day 17 after therapy administration), but decreased in size again over the course of the experiment. A similar tumour growth inhibition pattern was found in mice intratumourally injected with [225Ac]AcDOTA; 6/8 tumours with inhibition of tumour growth (Fig. 5B). Five out of eight tumours doubled in size during the experiment (mean time after treatment administration was 67 ± 31 days), but four of these tumours decreased again during the study. Tumours in the two control groups (no intratumoural injection and tumours injected with PBS) showed a pronounced growth starting around 50 days after tumour inoculation (Fig. 5C,D). Seven out of eight tumours were doubled in size at an average of 35 ± 10 days after PBS administration. Six out of eight tumours of the non-injected mice did not show tumour growth inhibition at the end of the study. The average tumour doubling time was 18 ± 9 days after treatment administration, but two of these tumours decreased in size over the course of the experiment. The PBS injection is not expected to have any influence on tumour growth, though the injection itself could have damaged the tumour tissue resulting in regression of the tumour. Surprisingly, tumour regression was also observed in some of the animals which had not received any treatment at all (Fig. 5D), showing that some of these tumours may spontaneously regress. Despite the fact that some tumours from the control groups also showed regression, we could still demonstrate that [225Ac]Ac-polymersomes significantly inhibited tumour growth as compared to the control groups. When time to tumour related decease is displayed in a Kaplan-Meier survival curve (Fig. 5E), the therapeutic efficacy of [225Ac]Ac-polymersomes and [225Ac]AcDOTA administration is evident as demonstrated by significantly (p < 0.05) improved survival as compared with the control groups.

The biodistribution study (Fig. 4) showed that [225Ac]Ac-polymersomes are retained very well at the tumour site, whereas [225Ac]AcDOTA is cleared rapidly. The tumour dose induced by the 50 kBq [225Ac]Ac-polymersomes is roughly equivalent to the dose given by 5 kBq 225Ac in polymersomes to a 400 µm tumour spheroid as studied earlier29 taking into account the polymersome uptake in the spheroids of only 0.10–0.15%. There, a significant reduction in tumour size was observed at 3–6 days after addition of the vesicles. In the current in vivo study, no significant increase in tumour size was observed after the injection of the polymersomes. The excellent response in the group injected with 50 kBq [225Ac]AcDOTA, which, similarly to the [225Ac]Ac-polymersome group, experienced no tumour related death over the treatment period, was somewhat unexpected. The biodistribution studies at 1 day p.i. show that at this point nearly all [225Ac]AcDOTA has been cleared from the tumour site (Fig. 4). However, in previous in vitro studies, [225Ac]Ac-polymersomes with an activity of only 0.1 kBq, already showed a decrease in tumour growth, indicating that very low amounts of activity can already influence tumour growth29. The dose received within the first few hours after injection of [225Ac]AcDOTA was thus sufficient to delay tumour growth, illustrating the effectiveness of α radionuclide therapy.

### Immunohistochemistry

To determine the effect of the intratumoural injection of the [225Ac]Ac-polymersomes, [225Ac]AcDOTA, as well as PBS on the tumour tissue, tissue sections were stained with HE and γ-H2AX (Fig. 6). The HE histological stain was used to demonstrate different tissue structures. While hematoxylin stains nuclei blue, eosin stains the cytoplasm as well as tissue fibers and matrigel36. Representative tumour sections of each of the treated groups are presented in Fig. 6. To detect the presence of double-stranded breaks (DSBs) in the DNA, γ-H2AX staining was used. γ-H2AX foci in the tumours gives an indication of the distribution and effectiveness of the α therapy, as the high LET of α radiation causes double strand breaks in DNA37. The tumours visualised in Fig. 6 show a similar uniform distribution of γ-H2AX foci in both the PBS and [225Ac]AcDOTA groups. The images show that the [225Ac]Ac-polymersomes treatment groups demonstrate a larger degree of γ-H2AX foci than the control groups, with an increasing foci gradient towards the centre of the tumour especially at day 1. As the polymersomes were injected in the centre of the tumour, the largest amount of radiation damage logically occurs in the vicinity of the centre. At 7 days p.i. the vesicles have not yet uniformly distributed themselves throughout the tumour, which is in line with the distribution rate as observed in the 3D tumour spheroids29, and is expected to improve at later time-points. However, despite the observed therapeutic efficacy in the [225Ac]AcDOTA group, the increase in DSBs was less pronounced. This could be due to the fast clearance of the [225Ac]AcDOTA complex from the tumour, and a subsequent decline in γ-H2AX signal to detect the DSBs. While the maximum number of γ-H2AX foci is reached 30 min after irradiation, the signal subsequently continuously decreases, reaching half their maximum already 2.5 h p.i.38,39. Hence, all damage to the tumour caused by [225Ac]AcDOTA will likely had happened within the first few hours p.i., whereas the treatment with [225Ac]Ac-polymersomes clearly irradiated the tumour over an extended period of time. Renal toxicity was not observed in any of the treatment groups, confirming the hypothesis that the amount of free 213Bi accumulated in the kidneys was not sufficient to cause long-term toxicity issues.

## Conclusion

The main objective of this paper was to study the distribution of free 213Bi in vivo after either intravenous or intratumoural injection of 225Ac encapsulated in polymersomes. It is well known that the recoil energy of the daughter nuclide upon α decay is sufficient to break any chemical bond, leading sometimes to renal toxicity caused by accumulation of 213Bi when using conventional antibody or peptide targeting approaches. Polymersomes have been shown to retain 225Ac daughter radionuclides to some extent21, and in this study we specifically studied the 213Bi retention and free 213Bi distribution in different tissues of mice. Two types of polymersomes were examined upon intravenous injection, where 225Ac was either encapsulated through conjugation with DTPA, or by coprecipitation with InPO4 nanoparticles contained within polymersomes. The benefit of the shorter recoil distance caused by the presence of the nanoparticle was shown, as more than twice the amount of 213Bi was retained in the case of the polymersomes containing nanoparticles. Upon intratumoural administration, 213Bi was retained in the tumour tissue to a large extend, with tumour: kidney ratios of 213Bi of 9.9 and 29.5 for 100 nm and 200 nm diameter polymersomes respectively. The therapeutic potential of intratumorally injected [225Ac]Ac-polymersomes was also studied, and although biodistribution studies showed very favourable retention of the polymersomes in the tumour tissue, whereas 225Ac coupled to DOTA was rapidly excreted, both formulations inhibited tumour growth. No tumour-related deaths were observed in either treatment group, and immunohistochemical analysis of the treatment groups at 1 d and 7 d p.i. showed an increase in γ-H2AX foci in the [225Ac]Ac-polymersomes group, indicating a larger degree of double-stranded breaks.

Concluding, while [225Ac]Ac-polymersomes and its daughters are retained at least partly the tumour site upon intratumoural administration showing their effectiveness in α therapy, their circulation time and tumour uptake upon intravenous administration needs further optimization before they can be used to target metastasized tumours. Furthermore, despite the much better retention of daughter nuclides in polymersomes compared to targeting molecules, 213Bi is still released and accumulates in the kidneys upon intravenous administration. This demonstrates how essential it is for any studies using 225Ac as α therapeutic to take daughter nuclide distribution into account.

## Methods

### Chemicals

The block copolymer PBd-PEO with Mw of 1900–900 g/mol was purchased from Polymer Source (Quebec, Canada). The 225Ac was obtained from the Directorate for Nuclear Safety and Security (Karlsruhe, Germany). The PD10 size exclusion columns were obtained from GE Healthcare (Hoevelaken, the Netherlands). Instant Thin-Layer Chromatography (iTLC) strips were purchased from Varian (USA). For the immunohistochemical analysis, rabbit-anti-H2AX (Cell Signaling, art.nr. 9718), goat-anti-rabbit (Vector, art.nr. BA-1000), avidin-biotin (Vectastain, art.nr. PK-6100) and Bright DAB (Immunlogic, art.nr. BSO4-500) were used. All other chemicals were purchased at Sigma Aldrich.

### DLS and Cryo-TEM measurements

Before being used for the in vivo studies, the vesicles were characterized by both dynamic light scattering (DLS) and cryogenic transmission electron microscopy (Cryo-TEM). The DLS consisted of a JDS Uniphase 633 nm 35 mW laser, a fibre detector, an ALV sp 125 s/w 93 goniometer and a Perkin Elmer photon counter, with an ALV-5000/epp correlator and software. A 0.01 mg/mL polymersome solution was placed in a toluene filled, temperature regulated bath (20 °C), where the intensity autocorrelation function was determined at 90°, and the data was fitted using the Contin method. The hydrodynamic radius was obtained using Einstein-Stokes equation.

Cryo-TEM images were obtained by depositing 4 μL of the 10 mg/mL polymersome solution on a holey carbon film (Quantifoil 1.2/1.3, Cu 200 mesh grids) supported on a TEM grid. The drop was blotted for four seconds with filter paper in order to obtain a thin layer on the grid, and subsequently vitrified by rapidly immersing in liquid ethane (Leica EM GP version 16222032). The sample was inserted into a cryo-holder (Gatan model 626) and then transferred to a Jeol JEM 1400 TEM. Images were obtained at an acceleration voltage of 120 keV. For the statistical analysis of the polymersome and nanoparticle diameters, about 30–50 images were made of each of the polymersome samples. The diameter of polymersomes and nanoparticles within those images were measured with ImageJ40.

For the radiolabelling of [225Ac]AcDOTA, 1.4 MBq 225Ac was added to 8 · 10−9 mol DOTA in 40 μL 0.1 M TRIS buffer at pH 9.0. After an incubation time of 1 h at 37 °C the labelling efficiency was determined with iTLC. A 2 µL droplet of the radiolabelled sample was placed on a 7 cm ITLC strip, with 0.1 M NaOH at pH 12 as mobile phase. The ITLC strip was subsequently imaged with the Phosphor-imager to determine the labelling efficiency, which was calculated as the fraction of the total activity which travelled with the mobile phase divided by the total activity on the sample. The sample was used without further purification, and diluted in PBS to an 225Ac concentration of 250 kBq/mL for intravenous, and 2 MBq/mL for intratumoural studies.

### Cell culture

The human breast cancer cell line MDA-MB-231 was cultured in RPMI-1640 (GIBCO, ThermoFisher Scientific, Waltham, MA, USA) media supplemented with 2 mM glutamine (GIBCO) and 10% fetal calf serum (Sigma-Aldrich Chemie BV), at 37 °C in a humidified atmosphere with 5% CO2. Cells were dissociated when 80–90% confluency was reached using 0.05% trypsin (w/v) in 0.53 mM EDTA (Life Technologies) and maintained as proliferating cultures. Mycoplasma contamination was evaluated every four months using a MycoAlert™ mycoplasma detection kit (Lonza, Basel, Switzerland). After thawing, cells remained in culture for a maximum of six months.

### Animal studies

All animal studies were approved by the Dutch central committee on animal research and the local ethical committee on animal research of the Radboud University under protocol 2015-0071, and performed according to the institutional guidelines. 6–8 weeks old female BALB/cAnNRj-Foxn1nu/Foxn1nu mice (Janvier Labs, France) were randomly tattooed for identification upon arrival. The mice were acclimatized for ≥4 days before any experimental procedure, and had unlimited access to food and water. Cages were replaced by clean cages every week, and the animals were housed with 5–6 mice per cage in a controlled environment (12 h dark/light cycle, 22 ± 1 °C, 55 ± 10% humidity). In all biodistribution studies, mice were sacrificed through CO2 asphyxiation and the organs of interest were collected, weighed, and the activity of two of the daughter nuclides of 225Ac, namely 221Fr (218 keV) and 213Bi (440 keV), were counted with the Wizard gamma counter (PerkinElmer) approximately 20 hours after sacrifice (when secular equilibrium was reached). The activity concentration for 225Ac was estimated from the daughter activity of 221Fr counted after equilibrium.

### Daughter nuclide retention upon intravenous injection

Retention of 213Bi in the polymersomes was determined in vivo through the intravenous injection of 200 μL of the 3 mg/mL polymersome solution containing 50 kBq 225Ac in the tail vain of five non-tumour bearing mice per group. Four hours post injection, the mice were sacrificed through CO2 asphyxiation. Organs of interest for daughter retention (blood, spleen and kidneys) were rapidly collected and the 213Bi activity was measured continuously on the Wizard gamma counter (PerkinElmer) for approximately 18 hours (Fig. 1). 213Bi activity at the time of sacrifice was determined by fitting the data with equation 1 and extrapolating back to t = 020:

$${A}_{2}(t)={A}_{1}(1-{e}^{-{\lambda }_{2}t})+{A}_{2}(0){e}^{-{\lambda }_{2}t}$$
(1)

where A1 the activity of 225Ac, and A2(t) is the 213Bi activity at time t with decay constant λ2. Figure 1 depicts the method of extrapolating the 213Bi activity in each of the three organs of interest at time of sacrifice. Paired two-sided Student’s t-tests were used to calculate the significance of the difference in organ uptake.

### Intratumoural injection

All mice used in the intratumoural experiments were subcutaneously inoculated with 5 · 106 MDA-MB-231 cells 1:1 in Matrigel. The tumour length, width and height were measured by calliper and the tumour volume was calculated using $${\rm{V}}=\frac{4}{3}\pi (\frac{W}{2})(\frac{L}{2})(\frac{H}{2})$$, with V the volume of the tumour, W the width, L the length and H the height of the tumour. Tumour size was used to block-randomized the mice over the different treatment groups. When the average tumour volume was approximately 100 mm3, the mice were intratumourally injected with either 25 μL, 50 kBq [225Ac]Ac-polymersomes, or 50 kBq [225Ac]AcDOTA, and one of the control groups with 25 μL PBS, while the other group was left untreated.

#### Biodistribution and recoil retention

The ex vivo biodistribution and 213Bi recoil retention were analysed at 1 and 7 days p.i. for the mice having been injected with [225Ac]Ac-polymersomes, or [225Ac]AcDOTA. The mice of the PBS and untreated groups were sacrificed for immunohistochemical analysis of the tumour and kidneys. Tumours and kidneys of all animals (3 per treatment group) were fixated in 4% paraformaldehyde in PBS overnight and embedded in paraffin to analyse double strand DNA breaks immunohistochemically. Retention of the daughter nuclide 213Bi in the tumour, and accumulation in the kidneys, was assessed according to the procedure in the intravenous experiment, where 213Bi activity at the time of sacrifice was determined by extrapolating the in-growth back to time t = 0.

#### Therapeutic evaluation

All groups consisted of 8 mice each, of which the tumour growth and overall survival was monitored up to 115 days after the start of treatment. Tumour volume was measured twice a week. Mice were taken out of the experiment when they reached one of the humane endpoints (tumour size > 2 cm3, ulceration or invasive tumour growth, other signs of clinical discomfort e.g. 15% weight loss in two days, dehydration). Technicians assessing the humane endpoints were blinded for the treatment groups.

### Immunohistochemistry

Tumours harvested at 1 and 7 days after the intratumoural injection were embedded in paraffin. Immunohistochemistry was performed using 4 µm thick tumour sections. For the haematoxylin and eosin (HE) staining, paraffin was removed by incubation with xylene, and subsequently sections were incubated with haematoxylin for 20 minutes and with eosin for 5 minutes, followed by dehydration and mounting in Permount.

For the staining for γ-H2AX, first antigen unmasking was performed on the deparaffinated tumour sections by treating the slides with 10 mM citrate buffer (pH 6.0) for 10 minutes at 98 °C. After washing the slides with distilled water and PBS, aspecific binding was blocked by incubating the sections with 50–150 µL 5% normal goat serum (NGS) in PBS for 30 minutes at RT. Subsequently, the tumour sections were incubated overnight with 50–150 µL rabbit-anti-H2AX diluted 1000 times in a PBS solution containing 1% BSA (bovine serum albumin) and 5% NGS at 4 °C. Subsequently, slides were washed 3x with PBS and endogenous peroxidase activity was blocked by a 10-minute incubation in 0.3% H2O2 in methanol at RT. After another two PBS washes, the sections were incubated for 30 minutes at RT with 200 times diluted biotinylated goat-anti-rabbit. Finally, an avidin-biotin complex was applied for 30 minutes at RT. To develop the tumour sections, they were incubated with 50–150 µL Bright DAB for 8 minutes at RT. Nuclei were counterstained with 3x diluted hematoxylin in PBS for 5 seconds. Finally, the sections were dehydrated with consecutively 50%, 70%, and twice 100% ethanol, twice with Xylene, after which they were mounted with Permount, dried and imaged.

### Statistics

Time-to event data collected during the intratumoural efficacy study were analysed using Graphpad Prism (version 5.03, Prism). Statistical significance between groups was analysed by multiple comparisons of the survival curves. Statistical significance was set at p < 0.05 calculated by the Log-rank Mantel-Cox test and corrected for multiple comparisons using a manually calculated Bonferroni-corrected threshold with 6 comparisons and p = 0.05.

## Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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## Acknowledgements

The present study was funded by the ﻿ SK foundation, the Zabawas foundation, and a ZonMw Early Career Scientist Hotel grant, project nr 43500401I.

## Author information

Authors

### Contributions

Conception, design of the work: R.M. de Kruijff, S. Heskamp, A.G. Denkova; collected data: R.M. de Kruijff, R. Raavé, A. Kip, J. Molkenboer-Kuenen; contributed data: A. Morgenstern, F. Bruchertseifer, analysed data: R.M. de Kruijff, R. Raavé, A. Kip, S. Heskamp, wrote paper: R.M. de Kruijff, reviewed and edited paper: R.M. de Kruijff, R. Raavé, A. Kip, J. Molkenboer-Kuenen, S. Heskamp, A.G. Denkova and A. Morgenstern.

### Corresponding author

Correspondence to R. M. de Kruijff.

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### Competing Interests

The authors declare no competing interests.

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Kruijff, R.M.d., Raavé, R., Kip, A. et al. The in vivo fate of 225Ac daughter nuclides using polymersomes as a model carrier. Sci Rep 9, 11671 (2019). https://doi.org/10.1038/s41598-019-48298-8

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