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Enabling Microparticle Imprinting to Achieve Penetration and Local Endurance in the Peritoneum via High-Intensity Ultrasound (HIUS) for the Treatment of Peritoneal Metastasis

Enabling Microparticle Imprinting to Achieve Penetration and Local Endurance in the Peritoneum... Hindawi International Journal of Surgical Oncology Volume 2020, Article ID 9679385, 7 pages https://doi.org/10.1155/2020/9679385 Research Article Enabling Microparticle Imprinting to Achieve Penetration and Local Endurance in the Peritoneum via High-Intensity Ultrasound (HIUS) for the Treatment of Peritoneal Metastasis 1 2,3 2 1 Agata Mikolajczyk, Tanja Khosrawipour, Alice Martino, Joanna Kulas, 1 1 4 2 Marek Pieczka, Maciej Zacharski , Jakub Nicpon, and Veria Khosrawipour Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Wroclaw, Lower Silesia, Poland Division of Colorectal Surgery, Department of Surgery, University of California Irvine (UCI), Orange, CA, USA Department of Surgery (A), University-Hospital Du¨sseldorf, Du¨sseldorf, North-Rhine Westphalia, Germany 1e Center of Experimental Diagnostics and Innovative Biomedical Technology, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland Correspondence should be addressed to Maciej Zacharski; maciej.zacharski@upwr.edu.pl Received 27 April 2020; Revised 6 July 2020; Accepted 11 August 2020; Published 25 August 2020 Academic Editor: Steven Curley Copyright © 2020 Agata Mikolajczyk et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Micro- and nanoparticles, with their submicron size, the versatility of physical and chemical properties, and easily modifiable surface, are uniquely positioned to bypass the body’s clearing systems. Nonetheless, two main problems with micro- and nanoparticles arise which limit the intraperitoneal application. *e study was performed to evaluate whether HIUS enables the imprinting of microparticles and, therefore, enhances penetration and local endurance in the peritoneum. Methods. High- intensity ultrasound (HIUS) at 20 kilohertz with an output power of 70 W was applied on peritoneal tissue samples from fresh postmortem swine for different time intervals. Before the HIUS application, the surface of the samples was covered with strontium aluminate microparticles before analysis via electron microscopy. In-tissue strontium aluminate penetration and particle dis- tribution size were measured using fluorescence microscopy on frozen thin sections. Results. With increasing HIUS durations (1 versus 5 minutes), increasing strontium aluminate particles were detected in the peritoneum. HIUS leads to a particle selection process with enhancing predominantly the penetration of smaller particles whereas larger particles had a harder time penetrating the peritoneum. Smaller particles were detected up to 277 µm ± 86 µm into the peritoneum. Conclusion. Our data indicate that HIUS might be used as a method to prepare the peritoneal tissue for micro- and nanoparticles. Higher tissue penetration rates without the increase and longer local endurance of the applied substance could be reached. More studies need to be performed to analyze the effect of HIUS in enhancing intraperitoneal drug applications. should remain active in the peritoneal cavity for an extended 1. Introduction period of time. Additionally, systemic absorption and tox- Peritoneal metastasis (PM) is a common manifestation of icity should be avoided. At the moment, most of the HIPEC, advanced gastrointestinal and gynecological cancers. *e PIPAC, and other forms of intraperitoneal chemotherapies cytostatics used for the treatment of PM do not remain in the are accomplished using the intravenous formulation of abdominal cavity for prolonged periods of time and are chemotherapeutic agents. Classic intraperitoneal chemo- instead quickly absorbed into the circulation due to the therapy drugs are susceptible to rapid clearance, exhibit local particularly small molecular weight of chemotherapeutics toxicity, and have limited penetration depths [3]. Nano- [1, 2]. For intraperitoneal administration, the ideal drug particles, with their submicron size, the versatility of physical 2 International Journal of Surgical Oncology and chemical properties, as well as easily modifiable surface, suspension was generated. For that, 500 mg luminescent are uniquely positioned to bypass the body’s clearing sys- particles were suspended in 3 ml of physiological saline tems. Nonetheless, two main problems with micro- and solution (0.9%). nanoparticles arise which limit the intraperitoneal 200 μL of luminescent particle suspension was dropped application. with a Pasteur pipette on the peritoneal surface which was *e first problem is that micro- and nanoparticles do not already covered by 5 mm of liquid. Next, high-intensity easily penetrate the peritoneal surface [4]. Fluid chemo- ultrasound (HIUS) was applied with a metal pen to the therapy does penetrate the peritoneum by molecular center of the peritoneal tissue using a sonicator (Sonoplus movement according to Fick’s law of diffusion. Although it is UW 2070, Bandelin, Berlin, Germany). *e tip of the pen known that the antitumor effect of intraperitoneal chemo- was within 3 mm of the tissue surface (Figure 1(a)). Samples therapy (IPC) is still strongly limited by the penetration of were divided into three groups which were treated for 0, 60, chemotherapy drugs less than 1 mm into peritoneal tissue and 300 seconds, respectively. Each treatment contained 0.3 [5, 6], there is at least some penetration into the target tissue. seconds of active and 0.7 seconds of passive interval, with 20 kHz frequency, output power of 70 W, and 50% of However, micro- and nanoparticles are not subjected to forces of diffusion. *ese particles do not penetrate the amplitude. peritoneal surface easily and thus are subject to drifting within the peritoneal cavity. Here, they accumulate on 2.2. Microscopic Analysis. After treatments, all tissues were certain hotspots and do not distribute evenly within the immediately frozen in liquid nitrogen. Cryosections (10 µm) peritoneal cavity [7–10]. *e problem with micro- and were prepared from different areas of each specimen. Sec- nanoparticles is that although they are not subject to rapid tions were mounted with ProLong Gold Antifade clearance like traditional chemotherapy drugs, they have Mountant (*ermo Fisher Scientific, Waltham, MA, USA) limited penetration into the peritoneum. Additionally, they containing 1.5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) do not evenly distribute within the peritoneum and, at to stain nuclei. *e penetration depth of luminescence par- present, are unable to target the peritoneal surface. Due to ticles was measured using the Nikon Eclipse 80i fluorescence their relatively large size in comparison with molecular microscope (Nikon Instruments Europe B.V. Amsterdam, chemotherapeutic agents, they are not governed by the Netherlands). *e distance between the luminal surface and forces of diffusion, and, therefore, it cannot be guaranteed the innermost positive staining for luminescence particles was that micro- and nanoparticles will have substantial inter- measured and reported in micrometers. action with the peritoneum than traditional chemothera- peutic agents. Manipulation of the chemical composition of these 2.3. Particle Detection on Scanning Electron Microscopy. particles has been attempted with the intention of signifi- A sample of the pestled luminescence surface particle was cantly increasing peritoneal residence time and prolonging placed on a glass-probe and was analyzed via scanning the exposure to chemotherapeutic agents. *is will also electron microscopy (SEM). Samples were spotted on alu- increase the local drug concentration, which is the primary minum tables, then dried, dusted with carbon (15 nm), and goal of intraperitoneal chemotherapy [11]. Ideally, the drug placed in the scanning chamber electron microscope (Auriga will be driven deeper into the peritoneal surface, increasing 60, Zeiss, Oberkochen, Germany). All samples were carried the time it remains within the peritoneum to also increase out at a beam voltage equal to 2 kV. *e luminescence the local drug concentration. We have termed this process particles within three cubic areas of 0.04 mm of the scans “imprinting.” Our study will analyze if HIUS could be used were subject to particle size measurements. to achieve “Imprinting” of solid micro- and nanoparticles into the deeper peritoneal tissue layer. 2.4. Ethical Approval and Regulations. Part of the experi- ments was performed on commercially available animal 2. Materials and Methods tissue samples. All methods were carried out in accordance with relevant guidelines and regulations which are applied 2.1. Peritoneal Tissue Model. *e experiments were per- according to the Polish law. Approval of the Local Board on formed on commercially available tissue samples. Fresh Animal Care was obtained (Zapytanie 8/8/2019) according postmortem swine peritoneum was purchased (local pork to Polish law. supplier, Zerniki Wielkie, Poland) and cut into proportional sections. Samples were then placed into Petri dishes, and NaCl 0.9% was added until the peritoneal surface was 2.5. Statistical Analyses. Experiments were independently covered with 5 mm of liquid. Luminescent particles were performed three times. A total of eight tissue sections per purchased in the form of powder (strontium aluminate tissue sample were subject to luminescence particle pene- powder, Sigma-Aldrich/Merck KGaA, Darmstadt, Ger- tration measurement. many). *e strontium aluminate powder was further ground For evaluating the distribution of the particle sizes, a with mortar to ensure that no residual large SA particles length of 200 µm of each tissue section (3 sections per remained. Part of these grounded particles was subject to sample) was subject to analyses. Prism 7.0 software electron microscopy for quality control and size measure- (GraphPad, La Jolla, CA, USA) was utilized to analyze the ments. As the strontium aluminate is not soluble in water, a data. Student’s t-test was used for the analyses of International Journal of Surgical Oncology 3 Distribution of particle size analyzed by electronmicroscopy High-intensity gh-intensity ultrasound pen rasound pen Luminescence particles Peritoneal surface 5 5–10 10–15 15–20 20–40 40–60 >60 Particle sizes in micrometer (µm) (a) (b) Figure 1: (a) Model of a high-intensity ultrasound directed (HIUS) “imprinting” of solid particles on the peritoneal surface. (b) Size distribution of luminescent particles. independent groups. A significant p value was considered at (60 seconds), and 277 µm ± 86 µm (300 seconds) (Figures 3 p< 0.05. and 4(a)). Penetration increased significantly with longer HIUS duration (0 seconds versus 60 seconds (p< 0.05) and 300 seconds p< 0.01) (Figure 4(a)). 161 luminescent par- 3. Results ticles (lp) were detected after 1 min of HIUS (111 lp with 3.1. Electron Microscopy of Luminescence Particles. *e <5 µm, 26 lp with 5–10 µm, 15 lp with 10–15 µm, 8 lp with electron microscopy analysis of the luminescence particles 15–20 µm, and no lp were larger than 20 µm) whereas 198 revealed a wide range of solid particle sizes. A total of 358 particles were measured after 5 minutes (113 lp with <5 µm, particles in the micrometer range were subject to size 42 lp with 5–10 µm, 23 lp with 10–15 µm, 16 lp with measurements. A large portion of the particles was around 15–20 µm, and 6 lp > 20 µm) (Figure 4(b)). *e number of 20–40 µm (Figures 1(b) and 2(a)). Noticeably smaller par- particles penetrating the peritoneum increased with de- ticles (<10 µm) were subject to particle electrostatic forces creasing particle diameter (Figure 4(b)). In particular, which accumulated these particles to conglomerates and particles less than 5 µm can be transported more easily clusters (2B). Smaller particles below 10 µm were, therefore, through the barrier. More than 50% of particles that pen- not observed as free particles. etrated the peritoneum were less than 5 µm (Figure 4(b)) after 60 seconds and 300 seconds of HIUS. However, as the duration of HIUS increased (300 seconds), larger particles 3.2. Ex Vivo Experiment. HIUS was applied without com- greater than 20 µm in size began to penetrate the plications. After applying HIUS, visual control of the sample peritoneum. was performed. However, after 300 seconds, some whitening and swelling of the peritoneum were noted. Luminescence particles were detected in fluorescence microscopy in all 4. Discussion three groups. Microscopic analysis of the different tissue specimens showed a substantial difference in the penetration Despite much progress in the development of antitumoral particles, their therapeutic applicability has been low. depth of the luminescence particles. Luminescence particles in the untreated samples remained on the peritoneal surface *ese particles seem promising in the treatment of PM due to their high antitumor potency and high cytotoxicity. and followed the surface terrain. Tissue penetration levels after HIUS were 42 µm ± 21 µm (0 seconds), 92 µm ± 42 µm However, their use is currently limited by their Percentual distribution of luminescence particle sizes (%) 4 International Journal of Surgical Oncology (a) (b) Figure 2: Electron microscopy analysis of solid luminescence particles (strontium aluminate). (a) Most particles’ sizes vary between <5 µm and 60 µm. (b) Particles smaller than 10 µm accumulate into clusters due to electrostatic effects and possibly temporarily disaggregate under high-intensity ultrasound. (a) (b) (c) Figure 3: Microscopic analysis of the penetration depth of luminescence particles into fresh peritoneal samples of Polish large white breed pigs. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI) intense white signal corresponding to the luminescence particles. Location of luminescence particles after (a) 0 seconds, (b) 60 seconds, and (c) 300 seconds of high-intensity ultrasound. distribution into the peritoneal cavity. *ese particles do Our data indicates that the pretreatment of tissue samples with HIUS enhances solid particle imprinting into not follow the same mechanics of standard liquid che- motherapeutic agents. Particles concentrate within dif- the peritoneal tissue. *is manipulation increases the local ferent body compartments, organs, and tissues [12, 13]. endurance of particles that would otherwise be washed away *is has been a significant problem in the application of or accumulate in other regions of the body. Furthermore, the these particles. For example, intraperitoneal application increased penetration depth reached by this method could of more complex particles such as liposomal doxorubicin improve antitumoral efficiency against peritoneal metastasis showed limited interaction with the surface and partial development. HIUS pretreatment has the potential to be a resorption [4, 14]. *e application of HIUS might, new approach for many forms of IPC. Yet, further research therefore, be a way to improve particle distribution and needs to be conducted for a translation of this ex vivo absorption in the peritoneal cavity. Direct imprinting as method into clinical practice. demonstrated might be an opportunity to place and en- A method to achieve a solution for both decreased sure the local endurances of these particles. tissue penetration and nonuniform particle distribution International Journal of Surgical Oncology 5 Particle penetration into the peritoneum aer In-tissue particle size aer high-intensity ultrasound high-intensity ultrasound aer 1 and 5 minutes ∗∗ 80 0 0 0 min 1 min 5 min <5 µm <10 µm <15 µm <20 µm >20 µm (a) (b) Figure 4: Microscopic analysis of the penetration depth of luminescence particles into fresh peritoneal samples of Polish white breed pigs. (a) In-tissue penetration of luminescence particles after 0, 1, and 5 minutes. (b) Particle sizes detected in the peritoneal according to particle size after 1 minute (yellow) and 5 minutes (red). may be a sort of quasi “Imprinting” of these particles into tissue by HIUS [23]. However, our study did not evaluate particle clearance or in-tissue endurance. In contrast to the peritoneal surface via high-intensity ultrasound (HIUS). *is could solve the problem of limited penetra- molecules and smaller nanoparticles, solid particles >1 µm are barely affected by diffusion forces and Brownian motion tion into the tissue by the relatively large particles in comparison to the molecular size chemotherapy. It would [24–26]. also prohibit the drifting of particles and, therefore, de- creasing the accumulation of particles on hotspots. Also, 5. Conclusion the agglomeration state of nano- and microparticles might significantly interfere with the biological uptake [15, 16]. HIUS could be a game-changer for micro- and nanoparticle Although the current research has been focused predom- IPC by improving the interaction of micro- and nano- inantly on its interference with the cellular uptake [17, 18], particles with the peritoneum. By increasing efficiency, local there might also be significant interference in regard to drug availability, and increased endurance of more complex biological surfaces. Although the forces in nanoparticle particles in the peritoneal cavity, HIUS has the potential to agglomeration are related to their surface energy, also significantly impact the utilization of micro- and nano- known as Van-der Waals force [19], the particle agglom- particles in the treatment of PM. In combination with new eration of solid microparticles is related to the electrostatic drug formulas and concepts, HIUS could enhance the ef- energy between them [20]. HIUS could be an option to ficiency of local drug delivery exceptionally and improve overcome these forces easily and, therefore, increase par- well-known limitations of local drug applications like lim- ticle interaction with the biological surface or possibly even ited penetration, limited endurance, and limited local greater cellular uptake of particles. concentration. It remains unclear whether particles are actively pushed into the cavities or sediments are brought into motion by Abbreviations HIUS and tear formation. It is also possible that both mechanisms play a role in particle penetration. While recent HIPEC: Hyperthermic intraperitoneal chemotherapy data on the effects of HIUS on the peritoneum have been HIUS: High-intensity ultrasound acquired, many technical aspects remain unclear. Another IPC: Intraperitoneal chemotherapy. possible advantage of HIUS pretreatment is the reduced absorption of particles through the lymphatic pathway. We Data Availability know that nano- and microparticles accumulate in the lymphatic system [21]. HIUS could also activate the particles *e data used to support the findings of this study are itself to interact with the surrounding surface and release available from the corresponding author on request. their chemical compounds as recently shown [13]. *e possibility of using HIUS to improve drug penetration of Conflicts of Interest fluid chemotherapy has already been demonstrated [22]. *is effect is supposedly attributed to the morphological *e authors have no conflicts of interest or financial ties to changes on the peritoneal surface and on the underlying disclose. Particle penetration in µm Percentual distribution of particle sizes 6 International Journal of Surgical Oncology carcinomatosis,” Drug Metabolism and Disposition, vol. 40, Authors’ Contributions no. 12, pp. 2365–2373, 2012. Agata Mikolajczyk and Tanja Khosrawipour contributed [10] A. Bellendorf, V. Khosrawipour, T. Khosrawipour et al., equally contributed to this work. AM contributed to study “Scintigraphic peritoneography reveals a non-uniform 99mTc-Pertechnetat aerosol distribution pattern for Pres- design, laboratory analysis, and data acquisition. TK con- surized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) in a tributed to the supervision of the study, drafting, and critical swine model,” Surgical Endoscopy, vol. 32, no. 1, pp. 166–174, revision for the important intellectual content of the manuscript; MA contributed to drafting and critical revision [11] P. H. Sugarbaker and O. A. Stuart, “Pharmacokinetics of the for the important intellectual content of the manuscript; JK intraperitoneal nanoparticle pegylated liposomal doxorubicin contributed to study design, laboratory analysis, data ac- in patients with peritoneal metastases,” European Journal of quisition, and manuscript drafting; MP contributed to data Surgical Oncology, vol. S0748–7983, no. 19, pp. 30380–4, 2019. interpretation and critical revision for the important in- [12] J. W. Nichols, Y. Sakurai, H. Harashima, and Y. H. Bae, tellectual content of the manuscript; MZ contributed to “Nano-sized drug carriers: extravasation, intratumoral dis- study design and critical revision for the important intel- tribution, and their modeling,” Journal of Controlled Release, lectual content of the manuscript; JN contributed to drafting vol. 267, pp. 31–46, 2017. and critical revision for the important intellectual content of [13] J. P. M. Almeida, A. L. Chen, A. Foster, and R. Drezek, “In the manuscript; VK contributed to study design, data vivo biodistribution of nanoparticles,” Nanomedicine, vol. 6, analysis, and manuscript drafting. no. 5, pp. 815–835, 2011. [14] A. Mikolajczyk, V. Khosrawipour, J Kulas et al., “Release of doxorubicin from its liposomal coating via high intensity Acknowledgments ultrasound,” Molecular and Clinical Oncology, vol. 11, no. 5, pp. 483–487, 2019. *is study was funded by institutional funds. [15] B. Halamoda-Kenzaoui, M. Ceridono, P. Urban ´ et al., “*e agglomeration state of nanoparticles can influence the References mechanism of their cellular internalisation,” Journal of Nanobiotechnology, vol. 15, no. 1, p. 48, 2017. [1] M. F. Flessner, “*e transport barrier in intraperitoneal [16] J. E. Skebo, C. M. Grabinski, A. M. Schrand, J. J. Schlager, and therapy,” American Journal of Physiology-Renal Physiology, S. M. Hussain, “Assessment of metal nanoparticle agglom- vol. 288, no. 3, pp. F433–F442, 2005. eration, uptake, and interaction using high-illuminating [2] R. L. Dedrick and M. F. Flessner, “Pharmacokinetic problems system,” International Journal of Toxicology, vol. 26, no. 2, in peritoneal drug administration: tissue penetration and pp. 135–141, 2007. surface exposure,” Journal of the National Cancer Institute, [17] A. Bruinink, J. Wang, and P. Wick, “Effect of particle ag- vol. 89, no. 7, pp. 480–487, 1997. glomeration in nanotoxicology,” Archives of Toxicology, [3] G. Los, E. M. E. Verdegaal, P. H. A. Mutsaers, and J. G. McVie, vol. 89, no. 5, pp. 659–675, 2015. “Pentetration of carboplatin and cisplatin into rat peritoneal [18] G. Pyrgiotakis, C. O. Blattmann, S. Pratsinis, and P. Demokritou, tumor nodules after intraperitoneal chemotherapy,” Cancer Chemotherapy and Pharmacology, vol. 28, no. 3, pp. 159–165, “Nanoparticle-nanoparticle interactions in biological media by 1991. atomic force microscopy,” Langmuir, vol. 29, no. 36, [4] A. Mikolajczyk, V. Khosrawipour, J. Schubert et al., “Effect of pp. 11385–11395, 2013. liposomal doxorubicin in pressurized intra-peritoneal aerosol [19] H. C. Hamaker, “*e London-van der waals attraction between chemotherapy (PIPAC),” Journal of Cancer, vol. 9, no. 23, spherical particles,” Physica, vol. 4, no. 10, pp. 1058–1072, 1937. pp. 4301–4305, 2018. [20] W. 1 Kaialy, “A review of factors affecting electrostatic [5] R. L. Dedrick, C. E. Myers, P. M. Bungay, and V. T. DeVita, charging of pharmaceuticals and adhesive mixtures for in- “Pharmacokinetic rational for the peritoneal drug adminis- halation,” International Journal of Pharmaceutics, vol. 503, tration in the treatment of ovarian cancer,” Cancer Treatment no. 1-2, pp. 262–276, 2016. Reports, vol. 62, no. 1, pp. 1–11, 1978. [21] Y. Xie, T. R. Bagby, M. Cohen, and M. L. Forrest, “Drug [6] G. Los, P. H. Mutsaers, W. J. van der Vijgh, G. S. Baldew, delivery to the lymphatic system: importance in future cancer P. W. de Graaf, and J. G. McVie, “Direct diffusion of cis- diagnosis and therapies,” Expert Opinion on Drug Delivery, diamminedichloroplatinum(II) in intraperitoneal rat tumor vol. 6, no. 8, pp. 785–792, 2009. after intraperitoneal chemotherapy: a comparison with sys- [22] V. Khosrawipour, S. Reinhard, A. Martino, T. Khosrawipour, temic chemotherapy,” Cancer Research, vol. 49, pp. 3380– M. Arafkas, and A. Mikolajczyk, “Increased tissue penetration 3384, 1989. of doxorubicin in pressurized intra-peritoneal aerosol che- [7] R. Liu, A. H. Colby, D. Gilmore et al., “Nanoparticle tumor motherapy (PIPAC) after high intensity ultrasound (HIUS),” localization, disruption of autophagosomal trafficking, and International Journal of Surgical Oncology, vol. 2019, Article prolonged drug delivery improve survival in peritoneal me- ID 6185313, 2019. sothelioma,” Biomaterials, vol. 102, pp. 175–186, 2016. [23] A. Mikolajczyk, T. Khosrawipour, J Kulas et al., “*e struc- [8] S. Hallaj-Nezhadi, C. R. Dass, and F. 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Dolez, Nanoengineering, Elsevier, Amsterdam, Nether- lands, 1st edition, 2015. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Surgical Oncology Hindawi Publishing Corporation

Enabling Microparticle Imprinting to Achieve Penetration and Local Endurance in the Peritoneum via High-Intensity Ultrasound (HIUS) for the Treatment of Peritoneal Metastasis

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Copyright © 2020 Agata Mikolajczyk et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi International Journal of Surgical Oncology Volume 2020, Article ID 9679385, 7 pages https://doi.org/10.1155/2020/9679385 Research Article Enabling Microparticle Imprinting to Achieve Penetration and Local Endurance in the Peritoneum via High-Intensity Ultrasound (HIUS) for the Treatment of Peritoneal Metastasis 1 2,3 2 1 Agata Mikolajczyk, Tanja Khosrawipour, Alice Martino, Joanna Kulas, 1 1 4 2 Marek Pieczka, Maciej Zacharski , Jakub Nicpon, and Veria Khosrawipour Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Wroclaw, Lower Silesia, Poland Division of Colorectal Surgery, Department of Surgery, University of California Irvine (UCI), Orange, CA, USA Department of Surgery (A), University-Hospital Du¨sseldorf, Du¨sseldorf, North-Rhine Westphalia, Germany 1e Center of Experimental Diagnostics and Innovative Biomedical Technology, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland Correspondence should be addressed to Maciej Zacharski; maciej.zacharski@upwr.edu.pl Received 27 April 2020; Revised 6 July 2020; Accepted 11 August 2020; Published 25 August 2020 Academic Editor: Steven Curley Copyright © 2020 Agata Mikolajczyk et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Micro- and nanoparticles, with their submicron size, the versatility of physical and chemical properties, and easily modifiable surface, are uniquely positioned to bypass the body’s clearing systems. Nonetheless, two main problems with micro- and nanoparticles arise which limit the intraperitoneal application. *e study was performed to evaluate whether HIUS enables the imprinting of microparticles and, therefore, enhances penetration and local endurance in the peritoneum. Methods. High- intensity ultrasound (HIUS) at 20 kilohertz with an output power of 70 W was applied on peritoneal tissue samples from fresh postmortem swine for different time intervals. Before the HIUS application, the surface of the samples was covered with strontium aluminate microparticles before analysis via electron microscopy. In-tissue strontium aluminate penetration and particle dis- tribution size were measured using fluorescence microscopy on frozen thin sections. Results. With increasing HIUS durations (1 versus 5 minutes), increasing strontium aluminate particles were detected in the peritoneum. HIUS leads to a particle selection process with enhancing predominantly the penetration of smaller particles whereas larger particles had a harder time penetrating the peritoneum. Smaller particles were detected up to 277 µm ± 86 µm into the peritoneum. Conclusion. Our data indicate that HIUS might be used as a method to prepare the peritoneal tissue for micro- and nanoparticles. Higher tissue penetration rates without the increase and longer local endurance of the applied substance could be reached. More studies need to be performed to analyze the effect of HIUS in enhancing intraperitoneal drug applications. should remain active in the peritoneal cavity for an extended 1. Introduction period of time. Additionally, systemic absorption and tox- Peritoneal metastasis (PM) is a common manifestation of icity should be avoided. At the moment, most of the HIPEC, advanced gastrointestinal and gynecological cancers. *e PIPAC, and other forms of intraperitoneal chemotherapies cytostatics used for the treatment of PM do not remain in the are accomplished using the intravenous formulation of abdominal cavity for prolonged periods of time and are chemotherapeutic agents. Classic intraperitoneal chemo- instead quickly absorbed into the circulation due to the therapy drugs are susceptible to rapid clearance, exhibit local particularly small molecular weight of chemotherapeutics toxicity, and have limited penetration depths [3]. Nano- [1, 2]. For intraperitoneal administration, the ideal drug particles, with their submicron size, the versatility of physical 2 International Journal of Surgical Oncology and chemical properties, as well as easily modifiable surface, suspension was generated. For that, 500 mg luminescent are uniquely positioned to bypass the body’s clearing sys- particles were suspended in 3 ml of physiological saline tems. Nonetheless, two main problems with micro- and solution (0.9%). nanoparticles arise which limit the intraperitoneal 200 μL of luminescent particle suspension was dropped application. with a Pasteur pipette on the peritoneal surface which was *e first problem is that micro- and nanoparticles do not already covered by 5 mm of liquid. Next, high-intensity easily penetrate the peritoneal surface [4]. Fluid chemo- ultrasound (HIUS) was applied with a metal pen to the therapy does penetrate the peritoneum by molecular center of the peritoneal tissue using a sonicator (Sonoplus movement according to Fick’s law of diffusion. Although it is UW 2070, Bandelin, Berlin, Germany). *e tip of the pen known that the antitumor effect of intraperitoneal chemo- was within 3 mm of the tissue surface (Figure 1(a)). Samples therapy (IPC) is still strongly limited by the penetration of were divided into three groups which were treated for 0, 60, chemotherapy drugs less than 1 mm into peritoneal tissue and 300 seconds, respectively. Each treatment contained 0.3 [5, 6], there is at least some penetration into the target tissue. seconds of active and 0.7 seconds of passive interval, with 20 kHz frequency, output power of 70 W, and 50% of However, micro- and nanoparticles are not subjected to forces of diffusion. *ese particles do not penetrate the amplitude. peritoneal surface easily and thus are subject to drifting within the peritoneal cavity. Here, they accumulate on 2.2. Microscopic Analysis. After treatments, all tissues were certain hotspots and do not distribute evenly within the immediately frozen in liquid nitrogen. Cryosections (10 µm) peritoneal cavity [7–10]. *e problem with micro- and were prepared from different areas of each specimen. Sec- nanoparticles is that although they are not subject to rapid tions were mounted with ProLong Gold Antifade clearance like traditional chemotherapy drugs, they have Mountant (*ermo Fisher Scientific, Waltham, MA, USA) limited penetration into the peritoneum. Additionally, they containing 1.5 µg/ml 4′,6-diamidino-2-phenylindole (DAPI) do not evenly distribute within the peritoneum and, at to stain nuclei. *e penetration depth of luminescence par- present, are unable to target the peritoneal surface. Due to ticles was measured using the Nikon Eclipse 80i fluorescence their relatively large size in comparison with molecular microscope (Nikon Instruments Europe B.V. Amsterdam, chemotherapeutic agents, they are not governed by the Netherlands). *e distance between the luminal surface and forces of diffusion, and, therefore, it cannot be guaranteed the innermost positive staining for luminescence particles was that micro- and nanoparticles will have substantial inter- measured and reported in micrometers. action with the peritoneum than traditional chemothera- peutic agents. Manipulation of the chemical composition of these 2.3. Particle Detection on Scanning Electron Microscopy. particles has been attempted with the intention of signifi- A sample of the pestled luminescence surface particle was cantly increasing peritoneal residence time and prolonging placed on a glass-probe and was analyzed via scanning the exposure to chemotherapeutic agents. *is will also electron microscopy (SEM). Samples were spotted on alu- increase the local drug concentration, which is the primary minum tables, then dried, dusted with carbon (15 nm), and goal of intraperitoneal chemotherapy [11]. Ideally, the drug placed in the scanning chamber electron microscope (Auriga will be driven deeper into the peritoneal surface, increasing 60, Zeiss, Oberkochen, Germany). All samples were carried the time it remains within the peritoneum to also increase out at a beam voltage equal to 2 kV. *e luminescence the local drug concentration. We have termed this process particles within three cubic areas of 0.04 mm of the scans “imprinting.” Our study will analyze if HIUS could be used were subject to particle size measurements. to achieve “Imprinting” of solid micro- and nanoparticles into the deeper peritoneal tissue layer. 2.4. Ethical Approval and Regulations. Part of the experi- ments was performed on commercially available animal 2. Materials and Methods tissue samples. All methods were carried out in accordance with relevant guidelines and regulations which are applied 2.1. Peritoneal Tissue Model. *e experiments were per- according to the Polish law. Approval of the Local Board on formed on commercially available tissue samples. Fresh Animal Care was obtained (Zapytanie 8/8/2019) according postmortem swine peritoneum was purchased (local pork to Polish law. supplier, Zerniki Wielkie, Poland) and cut into proportional sections. Samples were then placed into Petri dishes, and NaCl 0.9% was added until the peritoneal surface was 2.5. Statistical Analyses. Experiments were independently covered with 5 mm of liquid. Luminescent particles were performed three times. A total of eight tissue sections per purchased in the form of powder (strontium aluminate tissue sample were subject to luminescence particle pene- powder, Sigma-Aldrich/Merck KGaA, Darmstadt, Ger- tration measurement. many). *e strontium aluminate powder was further ground For evaluating the distribution of the particle sizes, a with mortar to ensure that no residual large SA particles length of 200 µm of each tissue section (3 sections per remained. Part of these grounded particles was subject to sample) was subject to analyses. Prism 7.0 software electron microscopy for quality control and size measure- (GraphPad, La Jolla, CA, USA) was utilized to analyze the ments. As the strontium aluminate is not soluble in water, a data. Student’s t-test was used for the analyses of International Journal of Surgical Oncology 3 Distribution of particle size analyzed by electronmicroscopy High-intensity gh-intensity ultrasound pen rasound pen Luminescence particles Peritoneal surface 5 5–10 10–15 15–20 20–40 40–60 >60 Particle sizes in micrometer (µm) (a) (b) Figure 1: (a) Model of a high-intensity ultrasound directed (HIUS) “imprinting” of solid particles on the peritoneal surface. (b) Size distribution of luminescent particles. independent groups. A significant p value was considered at (60 seconds), and 277 µm ± 86 µm (300 seconds) (Figures 3 p< 0.05. and 4(a)). Penetration increased significantly with longer HIUS duration (0 seconds versus 60 seconds (p< 0.05) and 300 seconds p< 0.01) (Figure 4(a)). 161 luminescent par- 3. Results ticles (lp) were detected after 1 min of HIUS (111 lp with 3.1. Electron Microscopy of Luminescence Particles. *e <5 µm, 26 lp with 5–10 µm, 15 lp with 10–15 µm, 8 lp with electron microscopy analysis of the luminescence particles 15–20 µm, and no lp were larger than 20 µm) whereas 198 revealed a wide range of solid particle sizes. A total of 358 particles were measured after 5 minutes (113 lp with <5 µm, particles in the micrometer range were subject to size 42 lp with 5–10 µm, 23 lp with 10–15 µm, 16 lp with measurements. A large portion of the particles was around 15–20 µm, and 6 lp > 20 µm) (Figure 4(b)). *e number of 20–40 µm (Figures 1(b) and 2(a)). Noticeably smaller par- particles penetrating the peritoneum increased with de- ticles (<10 µm) were subject to particle electrostatic forces creasing particle diameter (Figure 4(b)). In particular, which accumulated these particles to conglomerates and particles less than 5 µm can be transported more easily clusters (2B). Smaller particles below 10 µm were, therefore, through the barrier. More than 50% of particles that pen- not observed as free particles. etrated the peritoneum were less than 5 µm (Figure 4(b)) after 60 seconds and 300 seconds of HIUS. However, as the duration of HIUS increased (300 seconds), larger particles 3.2. Ex Vivo Experiment. HIUS was applied without com- greater than 20 µm in size began to penetrate the plications. After applying HIUS, visual control of the sample peritoneum. was performed. However, after 300 seconds, some whitening and swelling of the peritoneum were noted. Luminescence particles were detected in fluorescence microscopy in all 4. Discussion three groups. Microscopic analysis of the different tissue specimens showed a substantial difference in the penetration Despite much progress in the development of antitumoral particles, their therapeutic applicability has been low. depth of the luminescence particles. Luminescence particles in the untreated samples remained on the peritoneal surface *ese particles seem promising in the treatment of PM due to their high antitumor potency and high cytotoxicity. and followed the surface terrain. Tissue penetration levels after HIUS were 42 µm ± 21 µm (0 seconds), 92 µm ± 42 µm However, their use is currently limited by their Percentual distribution of luminescence particle sizes (%) 4 International Journal of Surgical Oncology (a) (b) Figure 2: Electron microscopy analysis of solid luminescence particles (strontium aluminate). (a) Most particles’ sizes vary between <5 µm and 60 µm. (b) Particles smaller than 10 µm accumulate into clusters due to electrostatic effects and possibly temporarily disaggregate under high-intensity ultrasound. (a) (b) (c) Figure 3: Microscopic analysis of the penetration depth of luminescence particles into fresh peritoneal samples of Polish large white breed pigs. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI) intense white signal corresponding to the luminescence particles. Location of luminescence particles after (a) 0 seconds, (b) 60 seconds, and (c) 300 seconds of high-intensity ultrasound. distribution into the peritoneal cavity. *ese particles do Our data indicates that the pretreatment of tissue samples with HIUS enhances solid particle imprinting into not follow the same mechanics of standard liquid che- motherapeutic agents. Particles concentrate within dif- the peritoneal tissue. *is manipulation increases the local ferent body compartments, organs, and tissues [12, 13]. endurance of particles that would otherwise be washed away *is has been a significant problem in the application of or accumulate in other regions of the body. Furthermore, the these particles. For example, intraperitoneal application increased penetration depth reached by this method could of more complex particles such as liposomal doxorubicin improve antitumoral efficiency against peritoneal metastasis showed limited interaction with the surface and partial development. HIUS pretreatment has the potential to be a resorption [4, 14]. *e application of HIUS might, new approach for many forms of IPC. Yet, further research therefore, be a way to improve particle distribution and needs to be conducted for a translation of this ex vivo absorption in the peritoneal cavity. Direct imprinting as method into clinical practice. demonstrated might be an opportunity to place and en- A method to achieve a solution for both decreased sure the local endurances of these particles. tissue penetration and nonuniform particle distribution International Journal of Surgical Oncology 5 Particle penetration into the peritoneum aer In-tissue particle size aer high-intensity ultrasound high-intensity ultrasound aer 1 and 5 minutes ∗∗ 80 0 0 0 min 1 min 5 min <5 µm <10 µm <15 µm <20 µm >20 µm (a) (b) Figure 4: Microscopic analysis of the penetration depth of luminescence particles into fresh peritoneal samples of Polish white breed pigs. (a) In-tissue penetration of luminescence particles after 0, 1, and 5 minutes. (b) Particle sizes detected in the peritoneal according to particle size after 1 minute (yellow) and 5 minutes (red). may be a sort of quasi “Imprinting” of these particles into tissue by HIUS [23]. However, our study did not evaluate particle clearance or in-tissue endurance. In contrast to the peritoneal surface via high-intensity ultrasound (HIUS). *is could solve the problem of limited penetra- molecules and smaller nanoparticles, solid particles >1 µm are barely affected by diffusion forces and Brownian motion tion into the tissue by the relatively large particles in comparison to the molecular size chemotherapy. It would [24–26]. also prohibit the drifting of particles and, therefore, de- creasing the accumulation of particles on hotspots. Also, 5. Conclusion the agglomeration state of nano- and microparticles might significantly interfere with the biological uptake [15, 16]. HIUS could be a game-changer for micro- and nanoparticle Although the current research has been focused predom- IPC by improving the interaction of micro- and nano- inantly on its interference with the cellular uptake [17, 18], particles with the peritoneum. By increasing efficiency, local there might also be significant interference in regard to drug availability, and increased endurance of more complex biological surfaces. Although the forces in nanoparticle particles in the peritoneal cavity, HIUS has the potential to agglomeration are related to their surface energy, also significantly impact the utilization of micro- and nano- known as Van-der Waals force [19], the particle agglom- particles in the treatment of PM. In combination with new eration of solid microparticles is related to the electrostatic drug formulas and concepts, HIUS could enhance the ef- energy between them [20]. HIUS could be an option to ficiency of local drug delivery exceptionally and improve overcome these forces easily and, therefore, increase par- well-known limitations of local drug applications like lim- ticle interaction with the biological surface or possibly even ited penetration, limited endurance, and limited local greater cellular uptake of particles. concentration. It remains unclear whether particles are actively pushed into the cavities or sediments are brought into motion by Abbreviations HIUS and tear formation. It is also possible that both mechanisms play a role in particle penetration. While recent HIPEC: Hyperthermic intraperitoneal chemotherapy data on the effects of HIUS on the peritoneum have been HIUS: High-intensity ultrasound acquired, many technical aspects remain unclear. Another IPC: Intraperitoneal chemotherapy. possible advantage of HIUS pretreatment is the reduced absorption of particles through the lymphatic pathway. We Data Availability know that nano- and microparticles accumulate in the lymphatic system [21]. HIUS could also activate the particles *e data used to support the findings of this study are itself to interact with the surrounding surface and release available from the corresponding author on request. their chemical compounds as recently shown [13]. *e possibility of using HIUS to improve drug penetration of Conflicts of Interest fluid chemotherapy has already been demonstrated [22]. *is effect is supposedly attributed to the morphological *e authors have no conflicts of interest or financial ties to changes on the peritoneal surface and on the underlying disclose. Particle penetration in µm Percentual distribution of particle sizes 6 International Journal of Surgical Oncology carcinomatosis,” Drug Metabolism and Disposition, vol. 40, Authors’ Contributions no. 12, pp. 2365–2373, 2012. Agata Mikolajczyk and Tanja Khosrawipour contributed [10] A. Bellendorf, V. Khosrawipour, T. Khosrawipour et al., equally contributed to this work. AM contributed to study “Scintigraphic peritoneography reveals a non-uniform 99mTc-Pertechnetat aerosol distribution pattern for Pres- design, laboratory analysis, and data acquisition. TK con- surized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) in a tributed to the supervision of the study, drafting, and critical swine model,” Surgical Endoscopy, vol. 32, no. 1, pp. 166–174, revision for the important intellectual content of the manuscript; MA contributed to drafting and critical revision [11] P. H. Sugarbaker and O. A. Stuart, “Pharmacokinetics of the for the important intellectual content of the manuscript; JK intraperitoneal nanoparticle pegylated liposomal doxorubicin contributed to study design, laboratory analysis, data ac- in patients with peritoneal metastases,” European Journal of quisition, and manuscript drafting; MP contributed to data Surgical Oncology, vol. 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International Journal of Surgical Oncology – Hindawi Publishing Corporation

Published: Aug 25, 2020

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