Available online at www.sciencedirect.
com
Minicells: Versatile vectors for targeted drug or si/shRNA cancer
therapy
Jennifer A MacDiarmid and Himanshu Brahmbhatt
Effective cancer therapy continues to be a daunting challenge
due mainly to considerable tumor cell heterogeneity, drug-
resistance, and dose-limiting toxicity of therapeutics. Here we
review a versatile nano-cellular (minicell) delivery vehicle that
can be packaged with therapeutically effective concentrations
of chemotherapeutic drugs, siRNAs or shRNAs and can be
targeted to tumors via minicell-surface attached bispecific
antibodies. A range of minicell-based therapeutics have shown
highly effective tumor stabilization/regression in the murine
xenograft model and in case studies in canines with late-stage
endogenous tumors. Repeat intravenous dosing shows
absence of toxicity or immunogenicity in both species. The
minicell-based therapeutic has potential applications in
personalized cancer medicine.
Address
EnGeneIC Ltd, Cancer Therapeutics, Building 2, 25 Sirius Road, Lane
Cove West, Sydney, NSW 2066, Australia
Corresponding author: Brahmbhatt, Himanshu
(hbrahmbhatt@engeneic.com)
Current Opinion in Biotechnology 2011, 22:909916
This review comes from a themed issue on
Pharmaceutical biotechnology
Edited by Luis Angel Fernandez and Serge Muyldermans
Available online 6th May 2011
0958-1669/$ see front matter
# 2011 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2011.04.008
Introduction
Cancer remains the major cause of death in most
advanced countries and the incidence of cancer increases
as populations age. Despite considerable advances in
development of cancer therapeutics, the disease con-
tinues to be plagued with a highly significant mortality
rate.
Chemotherapeutic drugs lack cancer-cell selectivity and
indiscriminate drug distribution results in severe toxicity
and limits anti-tumor efficacy. Owing to the intrinsic
genetic diversity and the ability to rapidly mutate, tumors
develop multi-drug resistance which contributes to the
high rate of treatment failure. To overcome these draw-
backs, efforts are being made to develop targeted drug
delivery systems such as stealth liposomes [1], micelles
www.sciencedirect.com
[2], nanoparticles [3,4], and polymerdrug conjugates
[5]. The very large number of different nanocarriers being
currently developed, several of which are FDA approved,
in clinical trials and marketed is reviewed [6]. However,
these technologies are also hampered by shortcomings,
such as drug leakage in vivo, lack of versatility in terms of
packaging a diverse range of different drugs without
significant derivatization, thereby reducing drug potency,
and difficulties in production scale-up, particularly for
nanoparticles.
Anti-cancer antibodies directed to over-expressed recep-
tors on cancer cells, such as the epidermal growth factor
receptor (EGFR; [7,8]) and HER2/neu [9] are less toxic,
but do not possess the potency and wide spectrum anti-
tumor activity of chemotherapeutic drugs. Additionally,
some for example HER2/neu are only over-expressed in
25% of breast cancer resulting in HER2/neu negative
breast cancer patients, meaning that the majority of
patients do not benefit from antibody therapy. The high
rate of mutations in the tumors also results in receptor
mutations [10] and consequent development of resist-
ance to antibody therapeutics.
In recent times, RNA interference (RNAi) has emerged
as a powerful technology for the development of cancer
therapeutics. RNAi is a natural phenomenon resulting in
potent post-transcriptional gene silencing produced by
double-strand RNAs that occur in most eukaryotes [11].
This multi-step process is initiated by double stranded
2030 nucleotide small interfering RNAs (siRNAs) or
microRNAs (miRNAs) resulting in highly efficient and
sequence-specific knockdown of the targeted genes
expression. siRNAs can also be expressed from plasmid
DNAs as short hairpin RNAs (shRNA) using an RNA
polymerase III promoter. RNAi has considerable poten-
tial in the treatment of cancer [12] with several ongoing
clinical trials. Nevertheless, several challenges need to be
overcome for exogenous siRNA to be widely used as a
cancer therapeutic. These include: firstly, lability of siR-
NAs, resulting in rapid degradation by serum nucleases,
secondly, poor membrane permeability of siRNAs limit-
ing cellular uptake, thirdly, need for effective design of
active siRNAs to ensure optimal gene silencing activity
with minimal off-target effects, and fourthly, need to
achieve efficient intracellular delivery to target cells in
vivo [13,14]. Several promising strategies have been
developed for systemic siRNA delivery such as nanopar-
ticles [15], aptamer-siRNA conjugates [16], nanoimmu-
noliposomes [17], cationic polymer and lipid-based
Current Opinion in Biotechnology 2011, 22:909916
910 Pharmaceutical biotechnology

siRNA complexes [18] etc., but several hurdles, including models, as well as in case studies of dogs with endogenous
delivery, still need to be overcome. tumors.

In this article we review a versatile tumor-targeted, nano-
cellular carrier, a bacterially derived minicell, capable of
carrying therapeutically significant concentrations of
drugs or siRNAs or shRNAs and can be targeted to tumors
via attachment of bispecific antibodies (BsAbs) to the
minicell-surface O-polysaccharide.
Bacterially derived minicells were first observed,
described, and termed minicells by Howard Adler
and colleagues in 1967 [19]. They are anucleate, non-
living nano-sized cells (400 nm in diameter) and are
produced as a result of mutations in genes that control
normal bacterial cell division [20,21,22] thereby de-
repressing polar sites of cell fission.
Previously we had reported that minicells can be pack-
aged with therapeutically significant concentrations of a
range of chemotherapeutics [23] or siRNAs and
shRNAs [24]. These minicells selectively targeted to
cancer cells via BsAbs, effect highly significant tumor
stabilization/regression in a variety of tumor xenograft
Packaging chemotherapeutic drugs or siRNAs
or shRNAs into minicells and tumor targeting
via attachment of bispecific antibodies
Minicells are derived from a minCDE-chromosomal
deletion mutant of Salmonella enterica serovar Typhimur-
ium (S. Typhimurium) and purified as described pre-
viously [23].
A range of chemotherapeutic and molecularly targeted
drugs with differing structure, charge, hydrophobicity,
and solubility such as doxorubicin, paclitaxel, irinotecan,
5-fluorouracil, cisplatin, carboplatin, gemcitabine, vin-
blastine, and monastrol, could be readily packaged within
the minicells ([23,24]; schematic shown in Figure 1).
This was accomplished by co-incubating each drug with
intact minicells for a period of time that was optimized for
each drug [23].
Drug-packaging in minicells was shown to be dependent
on both the concentration of drug in the loading solution,
and time of incubation [23]. Drug permeation into

Figure 1

Empty minicells Insertion of anti-cancer drug

OR

siRNAs

Attachment of
bispecific
antibody

Attachment to
cancer cell
surface receptor

Current Opinion in Biotechnology

Schematic showing bispecific antibody-targeted, drug/siRNA-packaged minicells. Schematic showing the packaging of anti-cancer drugs or siRNAs
into empty minicells and targeting them to tumor cell-surface receptor using bispecific antibodies where one arm of the antibody has minicell-surface
O-polysaccharide specificity and the other arm has specificity for the tumor cell-surface receptor for example EGFR.

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 Minicells as versatile vectors for targeted delivery MacDiarmid and Brahmbhatt 911

minicells possibly occurs via non-specific porin channels
[25] in the outer membrane. High resolution structural
analysis of some bacterial porins suggests that permeation
of chemicals through the porin channel is driven by
molecular interactions with the surface rather than by
free diffusion [26,27]. Detailed studies of porins have
revealed charged residues within the channels resulting
in a transversal electric field that separates polar and non-
polar solutes. Polar solutes are thought to be oriented in
the field during permeation which therefore becomes a
rapid one-dimensional entry process [28]. Nonspecific
movement of hydrophobic solutes across the outer mem-
brane possibly occurs through other channels such as the
FadL family of outer membrane proteins [29,30] and
OmpW [31]. Thus it is likely that drug entry into mini-
cells is through a facilitated entry process. Leakage of the
drug from the minicells was not observed following
incubation in buffer or serum for over 24 hours.
Quantitation of drug-packaged in minicells showed that
approximately a million molecules of doxorubicin were
loaded per minicell. In contrast, other nanoparticles such
as liposomes have been shown to package 10,000 mol-
ecules of drug per liposome [32]. Similarly, armed anti-
bodies can conjugate only less than 10 drug molecules per
antibody. The potency of anti-tumor effects may depend
on the concentration of a drug that is delivered intra-
cellularly within cancer cells.
siRNAs are also packaged in minicells through co-incu-
bation ([24]; schematic shown in Figure 1). shRNA
encoding plasmids are initially transformed into the min-
CDE-S. Typhimurium strain and the plasmid segregates
into the minicells. When the minicells are purified, they
are therefore pre-packaged with therapeutically signifi-
cant copies of the plasmid. PCR quantitation studies
showed that 50 copies of plasmid or 14,000 copies
of siRNA are packaged per minicell.
Targeting of minicells to tumor cells was achieved using
bispecific antibodies in which one arm recognizes the O-
polysaccharide component of the minicell surface lipo-
polysaccharide (LPS) and the other, a cell-surface re-
ceptor specific for the mammalian cell to be targeted,

Figure 2

Transport of antibody-targeted drug minicell attachment to tumor

or siRNA-packaged minicells cell surface (active targeting)

minicells fall out

of leaky vasculature
(passive targeting)

Current Opinion in Biotechnology

Schematic showing passive and active targeting to tumors in vivo. Schematic showing that post-i.v. administration of the minicell-based anti-cancer
therapeutic, the minicells accumulate in the tumor microenvironment due likely to the EPR effect (passive targeting) which is then followed by the
process of active targeting via attachment of the minicell-surface associated bispecific antibody to the tumor cell surface receptor for example EGFR
(depicted in Figure 3).

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912 Pharmaceutical biotechnology
for example EGFR [33] or HER2/neu receptor [34] on
breast and ovarian cancer cells, respectively. Linkage of
these two antibodies via their Fc regions was achieved
using protein A/G. This ensures absence of complement-
mediated in vivo toxicity since the Fc part of each
monoclonal antibody is blocked by protein A/G. The
tumor targeting antibody can be varied depending on
the desired tumor target.
Mechanism of passive targeting of solid
tumors post-i.v. administration of drug/si/
shRNA-packaged, BsAb-targeted minicells
Post-intravenous administration, the BsAb-targeted,
drug/siRNA/shRNA-packaged minicells appear to
rapidly fall out of the vascular circulation and into
the tumor microenvironment possibly due to the leaky
vasculature associated with solid tumors, a phenom-
enon recognized as the enhanced permeation and
retention effect (EPR; [35,36,37,38,6]; schematic
shown in Figure 2).
These investigators demonstrated that most solid
tumors have blood vessels with defective architecture
and usually produce extensive amounts of various vas-
cular permeability factors. Most solid tumors therefore
exhibit enhanced vascular permeability thus ensuring
nutrient and oxygen supply to tumor tissues. There is
general agreement that the fenestrations associated
with abnormal tumor vasculature can range from
10 nm to 1000 nm or more depending on the tumor
type, malignancy, and stage of disease [39,40,41].
Additionally, it is also recognized that proliferating
cancer cells in tumor tissue compress lymphatic vessels
particularly at the center of the tumor [42] resulting in
inefficient drainage of fluid from the tumor center and
this coupled with vascular content leakage from tumor
vessels causes interstitial hypertension which is thought
to reduce the delivery of therapeutic agents to solid
tumors [43,44].
Although the EPR effect has provided an excellent oppor-
tunity for the development of a large number of different
nanoparticle delivery systems for cancer therapy, in recent
times, new hurdles have been identified. For example, the
larger the tumor, the greater the pathophysiological hetero-
geneity and the central area of these tumors do not appear
to exhibit the EPR effect [45].
This unique phenomenon in solid tumors the EPR
effect is considered to be a landmark principle in
tumor-targeting chemotherapy and is becoming increas-
ingly important for anticancer drug development. For
example, Doxil1, a PEGylated (polyethylene glycol-
coated) doxorubicin-packaged liposome, DaunoX-
ome1, liposomal daunorubicin, and Abraxane1, a nano-
particle albumin-bound paclitaxel have all been
approved for treatment of several different cancers.
Current Opinion in Biotechnology 2011, 22:909916
Mechanism of intracellular delivery of drug/si/
shRNA via drug/si/shRNA-packaged,
BsAb-targeted minicells
Once in the tumor microenvironment, the BsAb-targeted
minicells actively target tumor cells via binding of the
tumor receptor-specific antibody to the tumor cell surface
receptor. In vitro studies demonstrated that the minicells
are endocytosed, degraded in late endosomes/lysosomes
and the payload (drug or siRNA or shRNA) is released
([23,24]; schematic shown in Figure 3). These studies
also showed that the payload appears to escape into the
tumor-cell cytoplasm in therapeutically significant concen-
trations although the mechanism of lysosomal membrane
escape is not understood. Interestingly, payloads like
shRNA encoding plasmids also escape the lysosomal mem-
brane and are translocated into the tumor cell nucleus
where shRNA expression occurs. In vivo xenograft studies
reveal that therapeutically significant concentrations of
shRNA are expressed in the tumor xenograft to enable
tumor stabilization and even regression. This has been
observed following the expression of shRNAs or delivered
siRNAs targeting mRNAs encoding multi-drug resistance
(MDR1, P-glycoprotein; [46]) and cell cycle-associated
proteins implicated in tumor cell proliferation, such as
polo-like kinase 1 (PLK1; [47]), kinesin spindle protein
(KSP; [48]) and cyclin-dependent kinase 1 (CDK1; [49]).
siRNAs or shRNAs delivered via BsAb-targeted minicells
to tumor cells were shown to effect potent G2 arrest and
apoptosis both in vitro and in vivo.
These cell-cycle associated proteins are of interest for
cancer therapy, since they are critically involved in cell
proliferation. Thus, PLK1 and CDK1 play an essential
role in the control of mitotic progression in proliferating
cells. KSP provides the propulsive forces required to
separate centrosomes during prophase, enabling them
to migrate to opposite poles and establish a functional
bipolar spindle.
Interestingly, chemotherapeutic payloads that produced
these potent anti-tumor effects were achieved with the
delivery of amounts of drug that are markedly smaller
than those required with systemic delivery of free drug.
For example, highly significant anti-tumor effects were
observed with 1875-fold and 8000-fold lower amounts
of Dox and Pac respectively delivered to xenografts via
minicells compared with the respective free drugs.
This was evident in dog case studies where rapid tumor
regression was evident in two dogs diagnosed with
advanced (stage IV) T-cell non-Hodgkins lymphoma
(NHL) when treated i.v. with anti-canine-CD3minicellsDox
[23]. One dog (4 kg) received a total of five doses over
35 days, and the other (40 kg), seven doses over 48 days
providing an average of 4.8 mg and 83.4 mg of Dox per dose
respectively. Interestingly, conventional chemotherapy in
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 Minicells as versatile vectors for targeted delivery MacDiarmid and Brahmbhatt 913

Figure 3

(a) Targeted minicell carrying therapeutic (b) minicells are swallowed

drug or siRNA binds to cancer cell by the cancer cell
surface receptor

(c) minicells broken down

B in the cancer cell

(d) Drug or siRNA escapes

E
from the minicell and
enters the cancer cell
(e) Tumor cell death
Current Opinion in Biotechnology

Schematic showing mechanism of intracellular delivery of drugs/siRNAs. Schematic showing the mechanism of minicell-mediated intracellular delivery
of the payload. The key steps are shown sequentially from A to E where (a) active binding of the minicell to the tumor cell-surface receptor via the
bispecific antibody, (b) endocytosis of the minicells by the tumor cells, (c) breakdown of the minicells in late endosomes/lysosomes, (d) release of drug
or siRNA into the vacuole and subsequent escape from the lysosomal membrane and into the cell cytoplasm, (e) tumor cell death following binding of
the therapeutic moiety to the respective intracellular target.

these dogs would require the administration of 8470 mg
and 39,300 mg of Dox per dose respectively (30 mg/m2) as
part of multi-drug combination chemotherapy. Thus the
treatment with CD3minicellsDox required 1764-fold and
471-fold less Dox per dose respectively, to achieve highly
significant tumor regression.
Treatment of drug resistant tumor via
sequential minicell-mediated delivery of
siRNA followed by drugs
Drug-resistance is a major limitation to effective long-
term cancer treatment [50]. Proof-of-concept studies in
vivo to treat drug-resistant cancers via minicell-delivered
therapies was demonstrated [24] using a dual sequential
treatment protocol in which the first treatment targets a
known drug resistance mechanism (e.g. overexpression of
the multi-drug resistance protein MDR1) via BsAb-tar-
geted, si/shRNA-packaged minicells. After allowing for
sufficient time to achieve highly significant knockdown of
the drug-resistance mediating protein, a second i.v.
administration is carried out with BsAb-targeted, cyto-
toxic drug-packaged minicells where the tumor was
known to be highly resistant to the cytotoxic drug. This
resulted in a dramatic reversal of drug resistance in vivo in
various xenograft murine models and even highly aggres-
sive multi-drug resistant uterine cancer xenografts were
eliminated with 100% survival in mice [24] treated with
the dual-minicell protocol. These results show that drug
resistance can be effectively reversed in previously highly
resistant tumor xenografts using minicell-mediated si/
shRNA delivery, and that such tumors are then exqui-
sitely sensitive to the second wave of minicells packaged
with the cytotoxic drug. This dual treatment strategy may
assist in the development of personalized treatment of
cancer particularly in the treatment of late-stage cancers
where current treatment options are seriously limited.
Biodistribution of minicells in mouse
xenograft studies
The biodistribution of i.v. administered 125I-labeled-mini-
cells in nude mice with EGFR overexpressing breast
cancer (MDA-MB-468) xenografts revealed that at two
hours post-treatment, 30% of the EGFRminicells were
localized in the tumor [23]. This was in contrast to 3% of
the anti-EGFR/O-polysaccharide BsAb that accumulated
in the tumor at two hours post-i.v. administration. By 6 and

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914 Pharmaceutical biotechnology
24 hours, 4.6% and 0.5%, respectively, of specifically
targeted EGFRminicells remained in the tumors. This data
suggested that the EGFRminicells rapidly extravasate from
the tumor-associated leaky vasculature and are trapped in
the tumor microenvironment likely due to the EPR effect.
Biodistribution of minicell-packaged drug following i.v.
administration of EGFRminicellsDox or free Dox to nude
mice with breast cancer xenografts (tumor volume be-
tween 140 and 170 mm3) showed that at six hours, 30%
of the Dox dose administered by the EGFRminicellsDox
was found in the tumors, as compared to only 1% of free
Dox.
Similar results were observed in mice with much larger
tumors (400600 mm3), in which 28.1% of the Dox dose
in EGFRminicellsDox was found in the tumors at six hours,
as compared to only 0.21% of that in non-targeted,
minicellsDox, and 1.8% for free Dox. Plasma concen-
tration of Dox at both time-points was undetectable.
At six hours, biodistribution to the liver, spleen, and lungs
was higher with the EGFRminicellsDox and minicellsDox
compared to free Dox and showed a rapid decrease by 24
hours.
Thus targeted minicell delivery provides at least a 30-fold
enrichment in tumor drug delivery.
Immune and cytokine/interferon response to
drug/si/shRNA-packaged, bispecific antibody
targeted minicells
In mouse xenograft studies there was no evidence of any
toxicity [23,24] despite repeat dosing of BsAb-tar-
geted, drug/si/shRNA-packaged minicells (1520 doses
have been administered in several mouse xenograft
experiments). This was evident by the lack of a febrile
response, weight loss, or skin/fur changes etc. in the
murine xenograft model. Importantly, minicells were well
tolerated with no adverse side-effects or deaths in any of
the treated animals.
Since minicells are of bacterial origin, it is necessary to be
cautious with systemic administration as bacterial pro-
ducts are known to elicit potent inflammatory responses
activated by Toll-like receptors [51]. A minicell purifi-
cation procedure to eliminate free endotoxin and free
bacterial components was developed to minimize the
potential for toxic side effects [23,24].
Recent studies have questioned whether the anti-tumor
effects of siRNA are due to specific knockdown of target
mRNA or, rather, are non-specific and merely due to
siRNA-mediated activation of the innate immune
response [52]. To address this issue, HCT116 xenograft
experiment was carried out [24] with mice being treated
with EGFRminicellssiPlk1, EGFRminicellssiNonsense or saline
Current Opinion in Biotechnology 2011, 22:909916
(controls). The treatments were administered four times,
24 hours apart. Groups of mice were sacrificed at 4 (early
response) and 24 (late response) hours post-treatment and
serum was collected and assayed for mouse and human
Type I and II interferons, and for inflammatory cytokines
produced by cells of the immune system. These cyto-
kines are known to be elicited following siRNA delivery
in mice [52].
The results showed that at both time points, human
interferon and cytokine levels were low (range of 10
to 30 pg/ml) and the levels elicited by EGFRminicells-
siPlk1 were indistinguishable from those observed with
saline or EGFRminicellssiNonsense. In contrast, mouse inter-
feron and cytokine responses were higher, being in the
range of 50 to 150 pg/ml for IFN-a, IFN-b and IFN-g,
300 to 1200 pg/ml for TNF-a, and 10 to 100 pg/ml
for IL-6. Both IFN-g and IL-6 showed a spike at four
hours but returned to baseline (saline control level) by 24
hours. Moreover, the TNF-a response was greater at 24
hours than at four hours. Importantly, however, there
were no significant differences in any of the interferon
or cytokine levels in mice treated with EGFRminicells-
EGFR
siPlk1, compared to minicellssiNonsense. The significant
rise in mouse TNF-a and IL-6 at 24 hours may be
attributed to the minicell vector itself since it is well
known that systemic administration of bacterial cells does
activate the Toll-like receptors resulting in TNF-a and
IL-6 responses. However, with minicell administrations
in mice, these responses have been self-limiting and have
not resulted in toxicity to the mice.
The above results indicate that the potent anti-tumor
effects observed in mouse xenografts are unlikely to be
due to interferon or inflammatory cytokine responses,
since treatment with EGFR-targeted siNonsense-pack-
aged minicells did not produce any anti-tumor effects
despite similar interferon and cytokine responses as those
in mice treated with EGFRminicellssiPlk1.
Conclusions
The minicell vector has quite remarkable properties in
being able to package, in therapeutically significant con-
centrations, a range of different drugs, siRNAs or shRNAs.
Each of these loadings is carried out following a simple co-
incubation of the therapeutic moiety with the intact mini-
cells in an appropriate buffer. The size of the minicell
(400 nm) ensures retention within the reticuloendothe-
lial system and prevents it from penetration into normal
tissues which is a hurdle faced by drug-conjugated mono-
clonal antibody therapeutics. For example, extravasation of
Cetuximab1 (anti-EGFR monoclonal antibody) into the
skin causes severe skin toxicity. The minicell vector is too
large to enter into normal skin, gastrointestinal or liver
tissue although these organs also exhibit leaky vasculature.
However, the fenestrations in these normal tissues range
from 50 nm to 100 nm, thus preventing minicell-based
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 Minicells as versatile vectors for targeted delivery MacDiarmid and Brahmbhatt 915
therapeutics from entering into these tissues. This was
demonstrated by MacDiarmid and colleagues in a rhesus
monkey trial (n = 36) where anti-monkey EGFR mono-
clonal antibody-targeted, doxorubicin-packaged minicells
were administered i.v. in monkeys in a dose-escalation
toxicology trial. Five repeat doses (one per week) were
administered. The results showed complete absence of
toxicity in the monkeys and inflammatory cytokine and
immune responses were also normal (unpublished data).
These results paved the way for the first-in-man, Phase I/
IIa multi-center clinical trial in cancer patients and this
trial is currently in progress.
Acknowledgement
We thank Martin Hale, Animated Biomedical Productions, Sydney, for the
artwork in Figures 13.
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