can you explain this in layman terms in detail : pharmaceutics
Article
The Feasibility of Pressurised Intraperitoneal Aerosolised
Virotherapy (PIPAV) to Administer Oncolytic Adenoviruses
Sophia J. Tate 1
, Leen Van de Sande 2
, Wim P. Ceelen 2
, Jared Torkington 3 and Alan L. Parker 1,*
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Citation: Tate, S.J.; Van de Sande, L.;
Ceelen, W.P.; Torkington, J.; Parker,
A.L. The Feasibility of Pressurised
Intraperitoneal Aerosolised
Virotherapy (PIPAV) to Administer
Oncolytic Adenoviruses.
Pharmaceutics 2021, 13, 2043.
https://doi.org/10.3390/
pharmaceutics13122043
Academic Editors:
Dragomira Majhen, Bozena
B. Michniak-Kohn, Bruno Sarmento
and Avi Domb
Received: 8 October 2021
Accepted: 25 November 2021
Published: 30 November 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1 Division of Cancer and Genetics, Cardiff University, Cardiff CF14 4XN, UK; <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>
2 Department of Human Structure and Repair, Ghent University, B-9000 Ghent, Belgium;
<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS> (L.V.d.S.); <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS> (W.P.C.)
3 Department of General Surgery, University Hospital of Wales, Cardiff CF14 4XW, UK;
<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>
* Correspondence: <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>
Abstract: Background: The prognosis of patients with peritoneal metastases is poor. Treatment
options are limited because systemically delivered chemotherapy is not usually effective in this type
of disease. Pressurised intraperitoneal aerosolised chemotherapy (PIPAC) is a recently developed
alternative technology for delivering intraperitoneal chemotherapy, potentially enhancing treatment
efficacy. Here, we assess the feasibility of pressurised intraperitoneal aerosolised virotherapy (PIPAV)
to deliver a different class of anticancer agents, oncolytic adenoviruses, in vitro and in vivo. Methods: Adenoviral vectors expressing reporter genes green fluorescence protein (Ad5.GFP) or firefly
luciferase (Ad5.Luc) were subject to pressurised aerosolisation. The ability of the virus to survive
PIPAV was assessed in vitro and in vivo by monitoring reporter gene activity. Wistar rats subjected to
PIPAV were assessed for any adverse procedure related events. Results: In vitro transduction assays
demonstrated that Ad5 retained viability following pressurised aerosolisation and could transduce
permissive cells equally effectively as non-aerosolised control vector. PIPAV was well tolerated in rats,
although minimal transduction was observed following intraperitoneal administration. Conclusions:
PIPAV appears viable and well tolerated, though in vivo efficacy requires further optimisation.
Keywords: oncolytic virus; delivery; peritoneum; PIPAC; PIPAV; peritoneal carcinoma; pressurised;
aerosolised; metastasis
1. Introduction
Malignant cells from an intra-abdominal primary tumour may invade the serosal
lining of the abdominal cavity causing peritoneal metastases (PM). PM are a late feature
of many cancers, making them common. They are challenging to treat, and prognosis is
generally poor. For example, the reported 5-year survival rate for patients with isolated
peritoneal metastases from colorectal cancer treated with systemic chemotherapy ranges
from 0 to 19% [1]. In patients with stage III ovarian cancer (i.e., spread to the abdominal
cavity) the average 5 year survival rate reported is 39% [1]. Thus, peritoneal metastases
represent an area of significant unmet clinical need.
A number of studies have demonstrated that patients with isolated peritoneal metastases have poorer survival outcomes than patients who have isolated metastases in solid
organs, such as the liver or lungs, if both groups are treated with systemic chemotherapy [2]. It is hypothesised that this is because the anatomical and physiological features
of the peritoneal mesothelium render it relatively resistant to systemically administered
therapeutics [3]. The capillary network supplying the mesothelial surface is located in a
submesothelial connective tissue layer, and it has been suggested that this acts as a ‘barrier’
to drug transport [3–5].
Intraperitoneal drug delivery has been suggested as a potential solution to this problem. Pressurised intraperitoneal aerosol chemotherapy (PIPAC) is a recent innovation for
Pharmaceutics 2021, 13, 2043. https://doi.org/10.3390/pharmaceutics13122043 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2021, 13, 2043 2 of 13
intraperitoneal administration of chemotherapy to treat peritoneal disease in advanced
malignancy [6]. During laparoscopic (keyhole) surgery, the abdominal cavity is inflated
with carbon dioxide, creating a pneumoperitoneum of 12 mmHg, to allow the surgeon
space to work. PIPAC involves aerosolising chemotherapy solutions into the abdominal
cavity once the pneumoperitoneum is established using a specially designed laparoscopic
nebuliser (Capnopen, Capnomed, Zimmern ob Rottweil, Germany). The aerosol is then
allowed to circulate in the pneumoperitoneum, which is maintained for 30 min. The
hypothesis behind PIPAC is that the pressure of the pneumoperitoneum counteracts the
tumour interstitial pressure, increasing absorption and penetration of drugs administered
into the abdominal cavity. Pre-clinical studies in animal models have demonstrated that
the distribution of solutions delivered using PIPAC is extensive and may be superior to
simple lavage of the cavity [7]. In vitro testing using human peritoneum demonstrated
increased depth of penetration of a fluorescent-labelled therapeutic agent applied using
PIPAC compared to the same agent applied using lavage [8]. The technique has been
used extensively to deliver chemotherapy in phase I/II trials, and the administration of
advanced therapeutics is now starting to be assessed in vitro and in vivo [9]. Thus far, the
delivery of nanoparticles [10] and messenger RNA [11] have been reported.
Adenoviruses have long been identified as having the potential to be effective anticancer agents. The structure and biology of human adenovirus serotype 5 (Ad5) has been
well described and understood [12]. Like other adenoviruses, they are non-enveloped, with
a double stranded DNA genome contained within an icosahedral capsid. They are obligate
intracellular parasites and can infect both dividing and non-dividing cells. The primary
receptor for Ad5 is the coxsackie and adenovirus receptor (CAR) [13]. They are lytic viruses,
causing destruction of the host cell on infection and release of progeny viruses into the
tissue. Thus, the therapeutic effect of an oncolytic virus can be amplified, since further cell
killing by progeny takes place following the cell killing from the initial infection of tumour
cells. Furthermore, it has been shown that the Ad5 genome can be manipulated by using
simple recombinant DNA techniques, to achieve high levels expression of foreign DNA
inserts [12]. High yields of virus can be generated and collected [12]. These properties are
all advantageous for an adenovirus-based cancer treatment and Ad5 has been a popular
vector choice for groups developing biological therapies as a result [14].
However, a potential limitation of Ad5 based vectors is that existing immunity is
common because of high seroprevalence rates across the world [15,16]. Studies have shown
that the humoral response to Ad infection generates antibodies targeted at epitopes on
multiple domains of the capsid. These antibodies act synergistically to neutralise the virus
on re-exposure [17]. This means that vectors may be neutralised by antibodies before they
reach their target tissue. Furthermore, it has been demonstrated that Ad5 forms complexes
with coagulation factor X in vivo to enter cells using heparan sulphate proteoglycans
(HSPGs), abundant on hepatocytes, as well as other cell types [18,19]. This results in rapid
sequestration of systemically administered Ad5 vectors by the liver and may impact on
uptake to other off target tissues following other routes of delivery.
One method of counteracting virus neutralisation is to administer the vector directly
into the target tissue, therefore enabling rapid entry of the virus to target cells and circumventing components of humoral immunity. The PIPAC technique potentially represents a
useful delivery method to enable delivery of oncolytic viruses directly to peritoneal metastases in the abdominal cavity. Intraperitoneal delivery would avoid interactions with blood
proteins and circulating antibodies that would occur as an intravenously administered
vector was delivered to the peritoneum in the blood.
One problem with direct administration to peritoneal metastases is that patients with
abdominal cancers frequently have ascites present in the peritoneal cavity, and this fluid
has also been shown to contain antibodies and immune cells that can neutralise adenovirus
vectors [20,21]. However, the ascites is removed at the start of a PIPAC procedure. Thus,
delivery by pressurised intraperitoneal aerosolisation may enable the provision of a more
favourable environment for the virus to bind and enter target cells. Our aim was therefore
Pharmaceutics 2021, 13, 2043 3 of 13
to assess the feasibility of pressurised intraperitoneal aerosolisation as a potential method
for the delivery of oncolytic adenovirus therapies to treat peritoneal metastases.
2. Materials and Methods
2.1. Viruses
The vectors used were based on an Ad5 genome captured in a bacterial artificial chromosome (BAC). Subsequent modifications were introduced into the BACs by homologous
recombineering [22]. Ad5 was engineered to express Green Fluorescent Protein (GFP) as a
reporter gene under the control of the short CMV IE promoter (Ad5.GFP), or to express
Luciferase as a reporter gene by the same method (Ad5.Luc). They had a complete E1/E3
gene deletion, rendering them replication deficient. Viruses were produced in HEK293
cells and purified and characterised according to standard protocols [23].
2.2. Cell Lines
A Wistar rat cell line (CC1, ECACC 93070901) was purchased from Public Health
England (Salisbury, UK). Chinese Hamster Ovarian Epithelial (CHO-K1) cells and Chinese
Hamster Ovarian transfected to express human CAR (CHO-CAR) were gifted by Dr Lynda
Coughlan.
2.3. In Vitro Assays
2.3.1. Effect of Aerosolisation
Cell lines were cultured and detached from the flasks when sub-confluent using 0.05%
trypsin. Complete medium was added to neutralise the trypsin, and then the cells were
counted using a haemocytometer. Cells were seeded in a flat bottomed 96 well plates at
20,000 cells per well and incubated overnight at 37 ◦C and 5% CO2.
Adenovirus vectors had been previously manufactured by homologous AdZ recombineering [22]. The Ad5.Luc and Ad5.GFP viruses were diluted in serum-free media (specific
to cell type) to a concentration of 2 × 109 vp/mL. Half of each solution was reserved
to carry out the control experiments. The remaining solutions were aerosolised using a
High-Pressure Injector (HPI) (Mark 7 Arterion Pedestal, Beyer, Leverkusen, Germany)
connected to a CapnoPen™ aerosoliser (Capnomed GmbH, Zimmern, Germany). The
aerosolised samples were collected in a clean 50 mL falcon tube.
The medium was removed from the wells of the 96 well plates and the cells were
washed with 200 µL PBS. The virus solutions were then diluted with appropriate serumfree media and added to the wells, with four replicates per condition (aerosolised and
non-aerosolized). A total of 100 µL of serum free medium was used as a negative control,
with four replicates per cell line. The plates were returned to the incubator for 3 h. The
medium was then removed from each well, the cells were washed with 200 µL of PBS, and
complete medium was added. The cells were incubated for a further 45 h. Expression of
the reporter genes was then assessed.
The expression of the luciferase reporter gene was assessed by Luciferase Assay
System (#1501, Promega UK Ltd., Southampton, UK) as per the manufacturer’s protocol.
The medium was removed from the wells and the cells were washed with PBS. Lysis
reagent (#E1531, Promega UK Ltd., Southampton, UK) was diluted in water as per the
manufacturer’s instructions and 100 µL was added to each well. The plates were frozen
at −80 ◦C. The plates were thawed, the contents of the wells mixed, and then 20 µL of
the lysate were transferred to a white 96 well plate. Then, 100 µL of luciferase reagent
were added to each well just prior to measurement of the luciferase activity in Relative
Light Units (RLU) on a multimode plate reader (Clariostar, BMG Labtech, Ortenberg,
Germany). The RLU was then normalised for total cellular protein (RLU/mg). The protein
concentration in each well was determined using a bicinchoninic acid (BCA) protein assay
(#23227; Pierce™ BCA Protein Assay Kit, Thermo Scientific, Loughborough, UK) as per the
manufacturer’s instructions. Bovine serum albumin (BSA) was used as a protein standard
at concentrations 2.0, 1.5, 1.0, 0.75, 0.5, 0.25, 0.125, 0.025, and 0 mg/mL in PBS. Next,
Pharmaceutics 2021, 13, 2043 4 of 13
10 µL of each standard, and 10 µL of each sample, were transferred to a 96 well plate in
duplicate. A total of 200 µL of working reagent (A:B = 50:1) was then added to each well,
and the plates were incubated for 30 min. The absorbance was measured at λ570 nm on an
iMark™ Microplate Absorbance Reader (BioRad, Hertfordshire, UK). The optical density
(OD) was normalised in all wells by subtracting the 0 mg/mL (PBS only) value from the
reading. A standard curve was prepared using the OD obtained from the BSA standards,
and the protein concentration in each well was deduced from the equation of the standard
curve. A two-way ANOVA with Tukey’s multiple comparison test was used to compare
the luciferase expression in each cell type and between those infected with aerosolised and
non-aerosolised virus.
The effect of different pressure conditions on the viability of Ad5.Luc was also assessed.
Plates were prepared in the same way, with CHO K1, CHO CAR, and CC1 cells plated at
20,000 cells per well and incubated for 24 h in complete medium. A solution of Ad5.Luc
in serum-free medium was made and serial dilutions carried out prior to application to
the cells in triplicate. The plates were placed in hyperbaric apparatus and the pressure
was maintained for 30 min. After 30 min, all plates were put in the incubator for a further
1
1
2
hours. The virus solution was removed and replaced with complete medium. After a
further 48 h incubation, the medium was removed and the cells were washed and frozen
in lysis buffer. The protein concentration in each well was determined using a BCA assay,
and the expression of luciferase by luminometry after the addition of luciferin as described
above. The mean RLU detected was normalised to protein concentration. A two-way
ANOVA with Tukey’s multiple comparison test was performed in GraphPad Prism 9.0 to
compare the results between the different pressure conditions.
The expression of GFP was assessed using flow cytometry. To prepare the cells, the
medium was removed from each well and the cells were washed twice with PBS. Trypsin
was then added to each well and the cells were incubated for 10 min. The trypsin was
neutralised by the addition of complete medium to each well, and then after mixing the
cells were transferred to a round-bottomed 96 well plate. The plate was spun at 1500 rpm
for 5 min. The supernatant was removed, and the cells were re-suspended in 100 µL of 2%
paraformaldehyde (PFA). The plate was left on ice for 15 min.
Once fixed, the samples were assessed on a BD Accuri C6 (BD Biosciences, Franklin
Lakes, NJ, USA) flow cytometer. A total of 10,000 events were recorded in channel FL-1.
Flow cytometry data were analysed using FlowJo v10 (FlowJo, BD Life Sciences, Ashland,
OR, USA). The negative control samples, which had only complete medium and no virus
applied, were used to gate the virus-exposed samples. The percentage of cells expressing
GFP was calculated, and multiple t-tests (GraphPad Prism, GraphPad Software Inc., La
Jolla, CA, USA) were used to compare the aerosolised samples with the non-aerosolised
samples.
2.3.2. In Vivo Studies
In vivo experiments were performed at the Laboratory of Experimental Surgery
(Ghent University, Ghent, Belgium). A rat model of PIPAC had already been developed
and used to test other therapeutics [24]. A series of small pilot experiments were designed
to assess the feasibility and tolerability of using the model to compare intraperitoneal injection of adenovirus vectors to intraperitoneal aerosolisation. The initial dose of Ad5.Luc
to be tested was selected based on previous experiments in rodent models. All animal
experiments were performed under approved protocols by the Animal Ethics Committee
of the Faculty of Medicine and Health Sciences, Ghent University, Belgium (ECD 17–109
and ECD 18–23), and in compliance with Belgian Council for Laboratory Animal Science
(BCLAS) guidelines for the Care and Use of Laboratory Animals.
Male Wistar rats (Envigo, Horst, The Netherlands) were used. The rats were allowed
to acclimatise to the surroundings for at least 48 h and were kept in standard housing
conditions with water and food at libitum and a 12 h light/dark cycle. The rats were
housed grouped by treatment allocation.
Pharmaceutics 2021, 13, 2043 5 of 13
In the first experiment, virus administration by intraperitoneal injection (group A)
was compared with intraperitoneal aerosolisation (group B). A dose of 3 × 1010 Ad5.luc
viral particles in 5 mL warmed 0.9% sodium chloride solution was used in both groups
(n = 3 per group), with negative controls (group C) receiving 0.9% sodium chloride solution
by intraperitoneal injection (n = 1) and aerosolisation (n = 1). The second experiment
was a dose escalation protocol. Intraperitoneal injection (group D) was compared with
intravenous injection (group E) in the first part of the study. A dose of 1 × 1011 vp
Ad5.Luc in 5 mL of warmed 0.9% sodium chloride solution was used in both groups
(n = 1 per group). Intraperitoneal injection of 0.9% sodium chloride solution was used as a
control (n = 1) (group F).
After intervention, the rats were observed for 72 h. Welfare observations were made
daily based on the rat grimace scale and body weight, and analgesia (Ketoprofen, 5 mg/kg,
SC) was provided as per the Animal House protocol. The rats were then imaged to determine the distribution of the Ad5.Luc by in vivo luminometry (IVIS Lumina II, PerkinElmer,
Zaventem, Belgium). Rats were anesthetised by inhalation of sevoflurane and injected with
1.5 mL of D-luciferin (PerkinElmer, Zaventem, Belgium) at 15 mg/mL into the peritoneal
cavity. Images were captured at 10 min. A one minute exposure was used in groups
A, B, and C, and a 10 min exposure was used in groups D, E, and F. Images were analysed by Living Image® software (PerkinElmer, Zaventem, Belgium) on a normalised scale
(photons/second/cm2/steradian). The rats were sacrificed with a lethal injection of T-61
(0.3 mL/kg, IV) into the tail vein and ex vivo luminometry of individual organs and tissues
was performed.
2.4. Statistical Analyses
Figures were created and statistical analyses performed with GraphPad Prism version
9.0 (GraphPad Software Inc., La Jolla, CA, USA). Unless otherwise stated, data show the
mean ± standard deviation (SD) or standard error of the mean (SEM) of n = 3–4 (specific n
numbers are indicated in each figure legend). p values were as follows: ns = not statistically
significant (p > 0.05); * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.
3. Results
3.1. Adenovirus Vectors Survive Aerosolisation and Retain Their Ability to Transduce Cells
Expressing Their Native Receptor In Vitro
To determine whether aerosolisation of adenoviral vectors using the CapnoPen device
negatively impacted on viral fitness, we performed transduction assays using aerosolised
and non-aerosolised Ad5.GFP (Figure 1). For these studies we utilised CHO CAR cells,
which express CAR, the native receptor for Ad5, and CHO K1, a cell line with no expression
of CAR, used for comparison. Cell receptor expression was confirmed using flow cytometry
(Figure 1a). Following infection of the CHO cells with aerosolised and non-aerosolised samples of Ad5.GFP, flow cytometry was carried out to compare the number of cells expressing
GFP from the non-aerosolised virus experiments versus the aerosolised virus experiments.
Gating for the flow cytometry was carried out using the control cell populations, which
were not exposed to virus, but maintained in complete medium. The equipment used
to aerosolise the virus solution is shown (Figure 1b), and flow cytometry data is shown
(Figure 1c). The results from the non-aerosolised virus experiments demonstrate that, as
expected, there was minimal expression of GFP in the transduced CHO K1 cells due to
the lack of CAR. The non-aerosolised Ad5 GFP was able to transduce CHO CAR cells in a
dose-dependent fashion. The same pattern of GFP expression was observed in samples
infected with aerosolised virus, suggesting that aerosolisation has no intrinsic effect on
the virus fitness. The proportion of CHO CAR cells expressing GFP in the non-aerosolised
versus the aerosolised samples was compared (Figure 1d) and there was no significant
difference in expression of GFP in cells inoculated with aerosolised virus when compared
to cells inoculated with non-aerosolised virus at any viral titre (1000 vp/cell p = 0.47,
2500 vp/cell p = 0.15, 5000 vp/cell p = 0.46).
Pharmaceutics 2021, 13, 2043 6 of 13 to transduce CHO CAR cells in a dose-dependent fashion. The same pattern of GFP expression was observed in samples infected with aerosolised virus, suggesting that aerosolisation has no intrinsic effect on the virus fitness. The proportion of CHO CAR cells
expressing GFP in the non-aerosolised versus the aerosolised samples was compared (Figure 1d) and there was no significant difference in expression of GFP in cells inoculated
with aerosolised virus when compared to cells inoculated with non-aerosolised virus at
any viral titre (1000 vp/cell p = 0.47, 2500 vp/cell p = 0.15, 5000 vp/cell p = 0.46).
(a) (b)
(c) (d)
Figure 1. Ad5.GFP survives aerosolisation using the CapnoPen device and retains its ability to transduce Wistar Rat
hepatocytes in vitro. (a) Flow cytometry data from immunocytochemistry to confirm receptor expression of CHO cells.
(b) Pictures to show the equipment used for the aerosolisation of the virus solution in vitro. (c) Flow cytometry data to
show the number of cells expressing GFP after infection with non-aerosolised (NAer) and aerosolised (Aer) vector. (d)
Quantification of CHO CAR cells expressing GFP after infection with non-aerosolised (NAer) and aerosolised (Aer) vector.
Error bars represent standard deviation;ns = not statistically significant (p > 0.05).
3.2. The Development of a Rat Model to Test Pressurised Intraperitoneal Aerosolisation of Adenovirus Vectors
Figure 1. Ad5.GFP survives aerosolisation using the CapnoPen device and retains its ability to transduce Wistar Rat
hepatocytes in vitro. (a) Flow cytometry data from immunocytochemistry to confirm receptor expression of CHO cells.
(b) Pictures to show the equipment used for the aerosolisation of the virus solution in vitro. (c) Flow cytometry data
to show the number of cells expressing GFP after infection with non-aerosolised (NAer) and aerosolised (Aer) vector.
(d) Quantification of CHO CAR cells expressing GFP after infection with non-aerosolised (NAer) and aerosolised (Aer)
vector. Error bars represent standard deviation; ns = not statistically significant (p > 0.05).
3.2. The Development of a Rat Model to Test Pressurised Intraperitoneal Aerosolisation of
Adenovirus Vectors
3.2.1. Adenovirus Vectors Are Able to Transduce Wistar Rat Hepatocytes In Vitro after
Aerosolisation Using the CapnoPen Device
An Ad5 vector with a firefly luciferase reporter gene (Ad5.Luc) was selected as the best
suited to an exploratory in vivo study, as it would enable luminometry by In Vivo Imaging
System (IVIS) (IVIS Lumina II, PerkinElmer, Zaventem, Belgium) imaging to be used assess
the expression of the reporter gene in the live animals. A preliminary experiment was
therefore performed to test the ability of Ad5.Luc to transduce rat hepatocyte CC1 cells
Pharmaceutics 2021, 13, 2043 7 of 13
in vitro after aerosolisation using the CapnoPen™ device (Capnomed GmbH, Zimmern,
Germany). CHO CAR and CHO K1 cells were again used as positive and negative controls.
The relative expression of luciferase in each cell line after transduction with aerosolised
and non-aerosolised virus was quantified (Figure 2a). There was no significant difference
in the RLU detected from cells incubated with aerosolised virus at a concentration of
10,000 vp/cell compared to those incubated with non-aerosolised virus at the same concentration. There was a significant difference in the mean RLU detected in the CC1 samples
compared to the CHO K1 samples, suggesting that the Ad5.Luc was able to infect and
transduce these cells, though not as efficiently as the CHO CAR cells.
Aerosolisation Using the CapnoPen Device
An Ad5 vector with a firefly luciferase reporter gene (Ad5.Luc) was selected as the
best suited to an exploratory in vivo study, as it would enable luminometry by In Vivo
Imaging System (IVIS) (IVIS Lumina II, PerkinElmer, Zaventem, Belgium) imaging to be
used assess the expression of the reporter gene in the live animals. A preliminary experiment was therefore performed to test the ability of Ad5.Luc to transduce rat hepatocyte
CC1 cells in vitro after aerosolisation using the CapnoPen™ device (Capnomed GmbH,
Zimmern, Germany).. CHO CAR and CHO K1 cells were again used as positive and negative controls.
The relative expression of luciferase in each cell line after transduction with aerosolised and non-aerosolised virus was quantified (Figure 2a). There was no significant difference in the RLU detected from cells incubated with aerosolised virus at a concentration
of 10,000 vp/cell compared to those incubated with non-aerosolised virus at the same concentration. There was a significant difference in the mean RLU detected in the CC1 samples compared to the CHO K1 samples, suggesting that the Ad5.Luc was able to infect
and transduce these cells, though not as efficiently as the CHO CAR cells.
(a)

✱✱
✱✱✱✱
Pharmaceutics 2021, 13, x FOR PEER REVIEW 8 of 14
(b)
Figure 2. Ad5.Luc survives aerosolisation using the CapnoPen device and retains its ability to transduce Wistar Rat hepatocytes in vitro under hyperbaric pressures. Quantification of luciferase expression in CHO K1, CC1, and CHO CAR cells following transduction with aerosolised Ad5.Luc
versus non-aerosolised Ad5.Luc versus control wells (a). Luciferase expression mediated by
Ad5.Luc at varying concentrations at under varying hyperbaric pressures of 20 mmHg; ns = not
statistically significant (p > 0.05); ** = p < 0.01; **** = p < 0.0001. (b). Error bars represent standard
deviation.
3.2.2. Adenovirus Vectors Are Able to Transduce Wistar Rat Hepatocytes In Vitro under
Atmospheric and Hyperbaric Conditions
Figure 2. Ad5.Luc survives aerosolisation using the CapnoPen device and retains its ability to
transduce Wistar Rat hepatocytes in vitro under hyperbaric pressures. Quantification of luciferase
expression in CHO K1, CC1, and CHO CAR cells following transduction with aerosolised Ad5.Luc
versus non-aerosolised Ad5.Luc versus control wells (a). Luciferase expression mediated by Ad5.Luc
at varying concentrations at under varying hyperbaric pressures of 20 mmHg; ns = not statistically
significant (p > 0.05); ** = p < 0.01; **** = p < 0.0001. (b). Error bars represent standard deviation.
Pharmaceutics 2021, 13, 2043 8 of 13
3.2.2. Adenovirus Vectors Are Able to Transduce Wistar Rat Hepatocytes In Vitro under
Atmospheric and Hyperbaric Conditions
Since delivery of therapeutics using pressurised intraperitoneal aerosolisation is performed under increased pressure, we sought to ascertain whether adenoviral vectors
retained complete ability to infect cells when placed under supraphysiological pressures.
We therefore assessed the ability of Ad5.Luc to transduce CC1 cells at a range of concentrations at atmospheric pressures or at hyperbaric pressures of 20 or 40 mmHg (Figure 2b).
The incubation of the cells with the virus solution was carried out in a pressurised chamber.
There was no significant difference observed between cells incubated at atmospheric pressure, 20 or 40 mmHg at any concentration. The pressures tested were higher than those
that would be used in the Wistar Rat model in vivo (i.e., 8 mmHg), providing comfort that
pressures used in vivo ought not to hamper adenoviral fitness.
3.2.3. Intraperitoneal Aerosolisation of an Adenovirus Vector Is Feasible and Is Tolerated
by Healthy Wistar Rats
Following the successful in vitro experiments, we next wished to test the feasibility of administration of virus vectors in an in vivo Wistar rat model. The pressurised
aerosolisation procedure was completed in four healthy Wistar rats (Group B and C2).
Following intervention, all rats were observed for 72 h. The rats who had undergone
the aerosolisation procedure had higher welfare scores (indicating greater distress) and
required analgesia on day 1 and day 2 post procedure. This was the case in both the rats
receiving adenovirus via aerosolisation, and the rat that received saline. Rats receiving
adenovirus by intravenous or intraperitoneal injection did not require analgesia. In vivo
imaging using the IVIS system was carried out 72 h after administration to assess the
level of transduction (Figure 3a). All the rats treated by intraperitoneal aerosolisation had
13 photons/second/cm2/steradian or less detected with no sign of localisation, indicating
it was likely to represent background signal.
Pharmaceutics 2021, 13, x FOR PEER REVIEW 9 of 14
(a)
(b)

Figure 3. Cont.
Pharmaceutics 2021, 13, 2043 9 of 13 Pharmaceutics 2021, 13, x FOR PEER REVIEW 10 of 14
(c)
Figure 3. Intraperitoneal administration of an adenovirus vector results in detectable transduction in a Wistar Rat model
and is well tolerated. IVIS imaging of rats at 72 h after intervention to determine the adenovirus vector biodistribution
after an intervention dose of Ad5.Luc 3 × 1010 vp (a). IVIS imaging of rats at 72 h after intervention to determine the
adenovirus vector biodistribution after an intervention dose of Ad5.Luc 1 × 1011 vp (b). Rat weights following intervention
(c). Weight loss >0 but ≤10% prompted administration of analgesia.
3.2.4. Intraperitoneal Administration of an Adenovirus Vector Results in Detectable
Transduction in a Wistar Rat Model
IVIS images were obtained after injection with the higher dose of virus (Figure 3b).
There was a greater signal seen with intravenous injection compared to intraperitoneal
injection, likely due to the high concentrations of blood clotting factor X in the blood which
can be engaged by Ad5 based virotherapies to efficiently enter cells presenting HSPGs.
The rats tolerated the increased dose well, with no increase in welfare scores and no requirement for analgesia.
4. Discussion
The feasibility of using pressurised intraperitoneal aerosolisation to administer oncolytic adenoviruses was investigated by preliminary work in vitro and in vivo using replication deficient Ad5 vectors expressing reporter genes. The in vitro experiments demonstrate that aerosolisation using the CapnoPen device does not affect the ability of Ad5-
based vectors to infect and transduce cells. Additionally, the virus is unaffected by exposure to hyperbaric conditions, and at pressures which exceeded those used clinically during PIPAC procedures was able to transduce cells efficiently. These findings were expected, since Ad5 causes respiratory illness and has therefore evolved to survive transmission by respiratory droplets. However, these are important findings in determining
whether intraperitoneal aerosolisation will be of use as a method of delivery for oncolytic
adenoviruses. Two Ad5 vectors were tested here; Ad5.GFP and Ad5.Luc. These differ
from vectors that would be used in a clinical context since they are non-replicating and
have relatively few modifications. This is a limitation of this study since these are not
Figure 3. Intraperitoneal administration of an adenovirus vector results in detectable transduction in
a Wistar Rat model and is well tolerated. IVIS imaging of rats at 72 h after intervention to determine
the adenovirus vector biodistribution after an intervention dose of Ad5.Luc 3 × 1010 vp (a). IVIS
imaging of rats at 72 h after intervention to determine the adenovirus vector biodistribution after an
intervention dose of Ad5.Luc 1 × 1011 vp (b). Rat weights following intervention (c). Weight loss >0
but ≤10% prompted administration of analgesia.
3.2.4. Intraperitoneal Administration of an Adenovirus Vector Results in Detectable
Transduction in a Wistar Rat Model
IVIS images were obtained after injection with the higher dose of virus (Figure 3b).
There was a greater signal seen with intravenous injection compared to intraperitoneal
injection, likely due to the high concentrations of blood clotting factor X in the blood
which can be engaged by Ad5 based virotherapies to efficiently enter cells presenting
HSPGs. The rats tolerated the increased dose well, with no increase in welfare scores and
no requirement for analgesia.
4. Discussion
The feasibility of using pressurised intraperitoneal aerosolisation to administer oncolytic adenoviruses was investigated by preliminary work in vitro and in vivo using
replication deficient Ad5 vectors expressing reporter genes. The in vitro experiments
demonstrate that aerosolisation using the CapnoPen device does not affect the ability of
Ad5-based vectors to infect and transduce cells. Additionally, the virus is unaffected by
exposure to hyperbaric conditions, and at pressures which exceeded those used clinically
during PIPAC procedures was able to transduce cells efficiently. These findings were
expected, since Ad5 causes respiratory illness and has therefore evolved to survive transmission by respiratory droplets. However, these are important findings in determining
whether intraperitoneal aerosolisation will be of use as a method of delivery for oncolytic
adenoviruses. Two Ad5 vectors were tested here; Ad5.GFP and Ad5.Luc. These differ from
vectors that would be used in a clinical context since they are non-replicating and have
relatively few modifications. This is a limitation of this study since these are not vectors
that would ultimately be used in the clinic. However, it is anticipated that more highly
engineered vectors based on Ad5 would tolerate aerosolisation equally well. One such
vector has been developed and described by Uusi-Kerttula et al. [23,25]. Ad5NULLA20 is
an Ad5 vector that has native tropisms ablated and has been retargeted to the tumourselective integrin αvβ6 through incorporation of an αvβ6-binding peptide (A20, NAVPNL-
Pharmaceutics 2021, 13, 2043 10 of 13
RGDLQVLAQKVART) within the fibre knob domain HI loop. This vector has been shown
to specifically infect epithelial ovarian cancer cells in vitro [23] and in vivo [21]. Further
investigation to test the ability of the Ad5nullA20 vector, and other specialised vectors,
to survive aerosolisation and retain their ability to specifically infect and kill target cells
would be an important area for future assessment of the feasibility of this technique in
clinical practice.
Administration to the local tumour environment using a technique such as PIPAV
may still be of benefit even when using a highly targeted vector because such vectors
can still be affected by neutralisation by the immune system. As already mentioned, the
ascitic fluid of patients previously exposed to adenoviruses can contain immune cells
and antibodies capable of neutralising a therapeutic vector [20,21]. The surgical part of
the PIPAV procedure in a human subject would allow drainage of the ascites, as well as
peritoneal lavage with warmed saline prior to administration of the therapeutic agent,
thus effectively removing and diluting factors which may negatively impact the efficacy
of oncolytic adenovirus. The direct administration of the vector to the peritoneal surface
using the nebuliser would potentially increase the concentration of the vector in direct
contact with the target tissue, and therefore the likelihood of virus binding and endocytosis
occurring before sequestration or neutralisation. It may also allow a lower dose overall
to be used, which might have positive implications for the safety and cost of treatment.
Further studies of adenovirus vectors in development using 3D tissue culture, and in the
presence of varying concentrations of ascites, would be useful to understand whether
greater tumour lysis occurs when ascites is removed in this way.
To ascertain whether the Wistar rat model would be an appropriate way to test
intraperitoneal aerosolisation of adenovirus vectors, experiments were performed in vitro
using a Wistar rat hepatocyte cell line (CC1). The Ad5 vector best suited for the rat model,
Ad5.Luc was able to transduce the Wistar rat cells in vitro with reasonable efficiency,
demonstrating that the rat cell line was able to be infected with a human adenovirus and
express a transgene. This did not translate into the expected results in the pilot experiment
in vivo using a dose of 3 × 1010 vp of Ad5.Luc. The dose selected was conservative when
compared with experiments previously carried out by the group in mice, but we were
conscious that the rats were going to undergo the aerosolisation procedure as well as
receiving the virus and did not want to induce dose-limiting toxicity. Additionally, we
discussed the proposed dose with other groups who used rat models to assess adenovirusbased vectors. The rats tolerated the procedure and the administration of the vector well.
However, transduction was not detected.
The second in vivo experiment therefore tested a higher dose of virus (1 × 1011 vp)
and compared transgene expression after IV and intraperitoneal delivery. The intraperitoneal route did result in transduction, though at a lower rate than IV delivery. This is
perhaps expected given previous research about the intraperitoneal route of administration
in humans [26] and other animal models [27,28] has also shown a lower uptake of therapeutic agents. The same rat model has been used to compare intraperitoneal injection,
intraperitoneal aerosolisation, and IV injection of mRNA complexes encoding the firefly
luciferase protein and found no significant difference in the average overall luminescence
detected from rats treated using each of the three administration routes [11]. The authors
commented that intraperitoneal aerosolisation and IV injection produced less variability
in the overall luminescence detected. There was a trend towards greater overall luminescence in the intraperitoneal injection group, but this was not significant because there
was more variability between rats treated using that route of administration. When they
looked at the distribution of the luminescence, the intraperitoneal aerosolisation technique
seemed to produce luminescence from a greater surface area of the abdomen of the imaged
rats, however, again, this was not a significant difference because of variability between
rats. Intraperitoneal injection and IV injection produced much less variable distribution,
with the signal concentrated to a smaller area in the upper abdomen. In this study using
adenovirus vectors, the high luminescence in the upper abdomen following intravenous
Pharmaceutics 2021, 13, 2043 11 of 13
delivery is likely to represent transduction in the liver of the rat. Ad5 forms complexes
with coagulation factor X to enter cells using heparin sulphate proteoglycans, and these
are abundant on hepatocytes [18,19]. We would not expect to see this pronounced effect
if an engineered vector was used as it is possible to ablate native tropisms of the virus to
achieve greater uptake in target tissues [21].
The detection of transduction in the second in vivo study, albeit at a lower level from
the intraperitoneal route of administration, supports further investigation in this rat model.
The rats did not appear to suffer any adverse consequences from the increased dose. These
results combined with the finding from the first in vivo study that the rats were able to
tolerate the intraperitoneal aerosolisation procedure using the lower dose of Ad5.Luc
support further investigation in this model. The absence of a dose escalation study in vivo
is a limitation of our work to date. An amendment to our ethics approval to perform the
second stage of the second in vivo study, comparing intraperitoneal aerosolisation and
intraperitoneal injection with an increased dose of 3 × 1011 vp Ad5.Luc was submitted and
approved in January 2020. Ultimately, the Severe Acute Respiratory Syndrome Coronavirus
2 pandemic prevented this experiment from going ahead as planned. This therefore remains
a promising avenue for future investigation.
5. Conclusions
We demonstrate that aerosolisation and hyperbaric pressure do not impact on the
fitness of adenoviruses, paving the possibility that such platforms could be amenable
to pressurised intraperitoneal aerosolised virotherapy applications in the future for the
treatment of peritoneal metastases. The in vivo feasibility study in Wistar rats demonstrated
the viability and tolerability of this approach, however, further studies at higher doses are
required to fully assess the efficacy of this approach for in vivo applications.
Author Contributions: Conceptualisation, S.J.T., J.T., A.L.P.; methodology, S.J.T., L.V.d.S.; formal
analysis, S.J.T.; investigation, L.V.d.S., S.J.T.; data curation, L.V.d.S., S.J.T.; writing—original draft
preparation, S.J.T.; writing—review and editing, S.J.T., L.V.d.S., A.L.P., J.T., W.P.C.; supervision, A.L.P.;
funding acquisition, S.J.T., A.L.P. All authors have read and agreed to the published version of the
manuscript.
Funding: S.J.T. was funded by a Wellcome Trust ISSF3 kickstart award to A.L.P. and S.J.T. (reference
513066).
Institutional Review Board Statement: The animal study was conducted according to the guidelines
of the Declaration of Helsinki and approved by the Animal Ethics Committee of Faculty of Medicine
and Health Sciences, Ghent University, Belgium (ECD 17–109 (approved 27 November 2017) and
ECD 18–53 (approved 2 July 2019)), and in compliance with Belgian Council for Laboratory Animal
Science (BCLAS) guidelines for the Care and Use of Laboratory Animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data presented in this study are available within the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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